Methods and reagents for efficient and targeted delivery of therapeutic molecules to CXCR4 cells

ABSTRACT

Conjugates comprising a targeting moiety specific for the CXCR4 and based on the polyphemusin-derived peptide and a therapeutic or imaging agent are provided. Therapeutic and diagnostic methods with the conjugates which require specific targeting to CXCR4+cells are provided as well.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/287,583, filed Feb. 27, 2019, which is acontinuation application of U.S. patent application Ser. No. 15/402,795,filed Jan. 10, 2017, now U.S. Pat. No. 10,265,371, issued Apr. 23, 2019,which is a continuation application of U.S. patent application Ser. No.13/979,560, filed Nov. 1, 2013, now U.S. Pat. No. 9,580,468, issued Feb.28, 2017, which is a U.S. national stage of International ApplicationNo. PCT/EP2012/050513 with the international filing date of Jan. 13,2012 which claims the priority benefit of the European PatentApplication No. 11382005 filed Jan. 13, 2011, the entire disclosures ofwhich are incorporated herein by reference.

FIELD OF INVENTION

The invention relates to conjugates comprising a targeting moiety and atherapeutic or diagnostic moiety as well as to the uses thereof fortherapy.

BACKGROUND OF INVENTION

Chemokine receptors are expressed on the surface of certain cells, whichinteract with cytokines called chemokines. The CXC chemokine receptor 4(CXCR4) is a G-protein-coupled receptor that transduces signals of itsendogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1,SDF-I). Following interaction of CXCR4/CXCL12, intracellular calcium(Ca²⁺) ions fluxes are triggered. This causes cellular responses,including chemotaxis, allowing cells to travel within the organism.

CXCR4 is expressed on myeloid cells, T-lymphocytes, B-lymphocytes,epithelial cells, endothelial cells and dendritic cells. Thus, theexpression of this molecule on the surface of tumor cells make it asuitable candidate as ligand for the specific targeting of therapeuticcompounds to cells expressing said molecule. For instance, WO2006029078describes fusion constructs comprising a protein translocation domainformed by the Tat protein, the CXCR4-receptor binding DV3 peptide domainand a therapeutic agent which is either a cdk2 antagonist peptide or ap53 activating peptide. These constructs are targeted to cellsexpressing CXCR4 and, by means of the protein translocation domain, thetherapeutic agent is translocated inside the cell. However, theseconstructs require, in addition to the CXCR4 ligand which acts solely inthe docking of the construct to the cell, a translocating domain whichdelivers the therapeutic agent inside the cell.

Driessen et al. (Molecular Therapy, 2008, 16:516-524) describes the useof a peptide analog,4-fluorobenzoyl-RR-(L-3-(2-naphthyl)alanine)-CYEK-(1-citrulline)-PYR-(1-citrulline)-CR(SEQ ID NO: 1), covalently linked to a phospholipid to target alipid-based gene delivery vehicle to CXCR4+-cells. However, this methodshows low efficency and requires increasing expression of CXCR4 on thesurface of the target cells by contacting the cells with VEGF prior tothe contacting with the conjugates.

Le Bon et al. (Bioconjugate Chem. 2004, 15, 413-423) describe the use ofthe CXCR4 specific ligands AMD3100 and AMD3100 for promoting specificgene transfer into cells expressing CXCR4 using lipid and polycationicconjugates. However, this method requires the conjugation of a phorbolester derivative to the polycationic lipid therapeutic agent in order toincrease CXCR4 expression on the surface of the target cells.

Egorova et al. (J Gene Med 2009; 11: 772-781) describe conjugates formedby CXCR4 ligands (the peptides KPVSLSYRSPSRFFESH-K9-biotin [(SEQ ID NO:2)-biotin], KPVSLSYR-K9-biotin [(SEQ ID NO: 3)-biotin] andD-LGASWHRPDK-K9-biotin [(SEQ ID NO: 4)-biotin] and DNA that binds thepolylysine region electrostatically and the use thereof for delivery ofnucleic acids to CXCR4 positive cells. However, these conjugates havelow efficiency and rely on the use of peptides showing agonisticactivity towards CXCR4 which may result in an increased tumorproliferation as a response of the stimulation of CXCR4 by the ligands.

Tamamura et al. (Bioinorganic & Medicinal Chemistry, 2001, 9: 2179-2187)describe conjugates of different analoges of the T140 CXCR4 agonist and3′-azido-3-deoxythymidine (AZT) and their use for preventingHIV-1-induced cytopathogenicity in MT-4 cells. However, no evidence wasprovided that these conjugates were capable of targetingCXCR4-expressing cells in vivo or that the AZT conjugated to the CXCR4ligand can be internalized by CXCR4.

Moreover, all these conjugates act by delivering the therapeutic agentto the surface of the cell from where internalization of the said agentrequires its binding to specific receptors on the cell surface.Therefore, there is a need in the art for further conjugates suitablefor the specific delivery of molecules of interest to CXCR4 cells whichovercome the problems of the conjugates described in the prior art andwherein internalization of the therapeutic agent occurs by the use of aCXCR4-targeting molecule which can be internalized by theCXCR4-expressing cells together with the therapeutic agent bound to it.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a conjugate comprising

-   -   (i) a targeting peptide comprising the sequence        RRWCYRKCYKGYCYRKCR (SEQ ID NO: 5) or a functionally equivalent        variant thereof and    -   (ii) a therapeutic agent        wherein the targeting peptide is capable of specifically binding        to CXCR4 and promoting internalization of the therapeutic agent        in a cell expressing CXCR4. In further aspects, the invention        relates to a polynucleotide encoding a conjugate according to        the invention, a vector comprising said polynucleotide and a        host cell comprising said polynucleotide or said vector.

In a further aspect, the invention relates to a conjugate, apolynucleotide, a vector or a host cell according to the invention foruse in medicine.

In a further aspect, the invention relates to a conjugate, apolynucleotide, a vector or a host cell according to the invention foruse in a method for the treatment of cancer wherein said cancer containscells that express CXCR4.

In a further aspect, the invention relates to a conjugate, apolynucleotide, a vector or a host cell according to the invention foruse in a method of treatment of a disease associated with HIV infection.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Internalization of T22, vCCL2, V1 and CXCL12-GFP-H6 fusionproteins in HeLa cells. Green fluorescent protein emission determined byflow cytometry, of untreated HeLa cells and 20 h after being exposed to0.6 μM of the CXCR4 ligand-GFP-H6 fusion proteins T22-GFP-H6, V1-GFP-H6,vCCL2-GFP-H6 and CXCL12-GFP-H6 in FIG. 1A PBS+10% glycerol buffer orFIG. 1B in Tris 20 mM+NaCl 500 mM buffer. HeLa cells had a prolongedtrypsin treatment (see experimental section) to eliminate potentialresidual fluorescent emission due to eventual cell surface attached GFPfusion proteins.

FIGS. 2A, 2B, 2C, 2D. Confocal analysis of T22, vCCL2, V1 andCXCL12-GFP-H6 upon internalization in HeLa cells. The cell membrane waslabelled with CellMask and cell DNA was labelled with Hoechst 33342.CXCR4 ligand-GFP-H6 fusion proteins produced a green signal that can beseen as intense dots inside the cells. Cultured cells were exposed for20 h to 0.6 μM of FIG. 2A T22-GFP-H6, FIG. 2B V1-GFP-H6, FIG. 2CvCCL2-GFP-H6 and FIG. 2D CXCL12-GFP-H6 proteins dissolved in PBS+10%glycerol buffer.

FIGS. 3A-3O. Characterization of T22-GFP-H6 entrance in HeLa cells. FIG.3A Green fluorescent protein emission determined by flow cytometry ofuntreated HeLa cells and 20 h after being exposed to differentconcentrations of T22-GFP-H6. FIG. 3B Green fluorescent protein emissiondetermined by flow cytometry of HeLa cells 20 h after exposure to 2 μMT22-GFP-H6, and with different trypsinization treatments previous tocytometry analysis. FIG. 3C Effect of serum on 2 μM T22-GFP-H6internalization measured by flow cytometry and analyzed by confocalmicroscopy in the presence of complete medium (FIG. 3E) or Optipro (FIG.3D), 20 h after protein addition. FIG. 3F-3H are isosurfacerepresentation of HeLa cells within a 3D volumetric x-y-z data fieldafter incubation with 2 μM T22-GFP-H6. FIGS. 3F-3H are TEM images ofrandomly T22-GFP-H6 particles in Tris 20 mM +NaCl 500 mM buffer. FIG. 3I2 μM T22-GFP-H6 internalization by confocal microscopy at 30 min (FIG.3J), 1 h (FIG. 3K), 2 h (FIG. 3L), 3 h (FIG. 3M), 4 h (FIG. 3N), and 24h (FIG. 3O).

FIGS. 4A-4D. Evaluation of DNA-binding and gene transfer properties ofT22-GFP-H6. FIG. 4A Retardation of plasmid DNA migration in agarose gelelectrophoresis promoted by increasing amounts of T22-GFP-H6. FIG. 4B,FIG. 4C and FIG. 4D Confocal microscopy of a HeLa cell with T22-GFP-H6protein inside (intense signal dots), which expresses td tomato gene(soft signal), 24 h after exposition to T22-GFP-H6-td tomato genecomplex.

FIG. 5 . Inhibition of T22-GFP-H6 internalization by a natural ligand ofCXCR4, the protein SDFalpha, at different T22-GFP-H6/SDFalpha ratios(1:0, 1:1, 1:10). Two irrelevant proteins, GFP-H6 and GLA, were used ascontrols at a 1:10 ratio.

FIG. 6 . Selective biodistribution of the T22-GFP, a fusion proteincomprising T22 as a target moiety (which specifically binds to the CXCR4receptor) and a green fluorescent protein, to primary tumor tissuederived from CXCR4+SW1417 human colorectal cancer cells xenotransplantedin the cecum of immunosuppressed mice. Notice the 8-47 fold accumulationof T22-GFP, as measured by fluorescence quantitation, using a IVIS200system (Xenogen) at a dose range varying form 20 to 500 ug, at severaltime points (5, 24 or 48 h), after intravenous single doseadministration. No fluorescence is detected in primary tumor tissue incontrol mice treated with vehicle. Fluorescence in the stomach is due toingested food, which does not differ between experimental and controlanimals. Black asterisk: Primary tumor.

FIG. 7 . Selective biodistribution of the T22-GFP to the peritonealmetastases and mesenteric and diaphragmatic lymph nodes derived fromCXCR4+SW1417 human colorectal cancer cells xenotransplanted in the cecumof immunosuppressed mice. Notice the 20-40 fold accumulation of T22-GFPin peritoneal metastatic foci, as measured by fluorescence quantitationusing a IVIS200 system (Xenogen) at a 500 ug dose at 5 h or 24 h afterintravenous single dose administration. No fluorescence is detected inthe liver parenchyma, pancreatic parenchyma, kidney, heart, non-tumorintestine, non-tumor lymph nodes, lung or spleen of the experimental orcontrol (vehicle control) animal. Whereas accumulation of the T22-GFPfusion protein is observed in tumor tissues, no accumulation of T22-GFPis observed in normal tissues. Fluorescence is observed in the billiaryvesicle (attached to the liver) or in the pancreas in both experimentaland control animals, which could be attributed to fluorescent proteinssecreted by these organs. Black asterisk: Primary tumor; Black arrow:Peritoneal metastasis.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have observed that, surprisingly, apeptide derived from the sequence of poliphemusin II([Tyr^(5,12),Lys⁷]-polyphemusin II, hereinafter T22 peptide) is able topromote the internalization of functional and soluble fused compoundsinto target CXCR4+ cells. As shown for instance in the examples of thepresent invention, a fusion protein comprising the T22 peptide and amarker protein can be internalized and released in the cytoplasm. Theresult was unexpected since, although this peptide had been reported inthe prior art as CXCR4 ligand (see Murakami et al. J. Virol., 1999,73:7489-7496), no increase in the internalization rates of the receptorwas reported as a result of the binding of the peptide to the receptor.The results were also surprising since the molecule that could beinternalized when coupled to the T22 peptide was a 26 kDa polypeptide.This type of molecule, due to their relatively large size, isinternalized with a much lower efficiency than small organic molecules.Moreover, the experiments provided in the present invention illustratethat other high-affinity CXCR4 ligands (the V1 peptide corresponding tothe N-terminal region of vCCL2, the complete vCCL2 or CXCL12/SDF-1),while capable of promoting endocytosis of the ligand, did not lead toendosomal escape of the proteins attached to said peptide.

Therapeutic Conjugates of the Invention

Thus, in a first aspect, the invention relates to a conjugate comprising

-   -   (i) a targeting peptide comprising the sequence        RRWCYRKCYKGYCYRKCR (SEQ ID NO: 5) or a functionally equivalent        variant thereof and    -   (ii) a therapeutic agent        wherein the targeting peptide is capable of specifically binding        to CXCR4 and promoting internalization of the therapeutic agent        in a CXCR4 expressing cell.

A. The Targeting Peptide

The first component of the conjugates of the invention is a targetingpeptide comprising the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 5)(hereinafter the T22 peptide) or a functionally equivalent variantthereof.

The term “peptide”, as used herein, generally refers to a linear chainof around 2 to 40 amino acid residues joined together with peptidebonds.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

The term “functionally equivalent variant”, as used herein, refers toany variant of the T22 which contain insertions, deletions orsubstitutions of one or more amino acids and which conservesubstantially the capacity of T22 for interacting with CXCR4 and forpromoting internalization and/or endosomal escape of therapeuticmolecules attached to it. A suitable assay for determining whether agiven peptide can be seen as a functionally equivalent variant thereofis, for instance, the assay described in example 1 of the presentinvention. In this assay, a putative functionally equivalent variant ofT22 variant is fused in frame with a marker polypeptide (e.g. afluorescent protein) and incubated with a cell expressing CXCR4 (e.g.HeLa cells). If the peptide is a functionally equivalent variant of T22,the marker protein will be internalized and expressed in the cytosol.

In one embodiment, the targeting peptide is the selected from the groupconsisting of:

-   -   the T140 peptide having the sequence RRX₁CYRKX₂PYRX₃CR (SEQ ID        NO: 6) wherein X₁ is L-3-(2-naphtyl)alanine, X₂ is D-Lys and X₃        is L-Citrulline.    -   the TN14003 peptide having the sequence RRX₁CYX₂KX₃PYRX₄CR (SEQ        ID NO: 7) wherein X₁ is L-3-(2-naphtyl)alanine, X₂ is Cit, X₃ is        dLys and X₄ is L-Citrulline,    -   the TC14012 peptide having the sequence RRX₁CYEKX₂PYRX₃CR (SEQ        ID NO: 8) wherein X₁ is L-3-(2-naphtyl)alanine, X₂ is        d-Citrulline and X₃ is L-Citrulline,    -   the TE14011 peptide having the sequence RRX₁CYX₂KX₃PYRX₄CR (SEQ        ID NO: 9) wherein X₁ is L-3-(2-naphtyl)alanine, X₂ is        L-Citrulline, X₃ is dGlu and X₄ is L-Citrulline and    -   the TZ14011 peptide having the sequence RRX₁CYX₂KX₃PYRX₄CR (SEQ        ID NO: 9) wherein X₁ is L-3-(2-naphtyl)alanine, X₂ is        L-Citrulline, X₃ is D-Lys and X₄ is L-Citrulline or the variant        thereof wherein the N-terminal Arginine residue is acetylated        (known Ac-TZ14011).

In another embodiment, the targeting peptide is not a peptide selectedfrom the group consisting of the T140 peptide, TN14003 peptide, TC14012peptide, the TE14011, the TZ14011 or the N-terminally acetylated variantof TZ14011 and the T131 peptide having the sequence RRYCYRKX₁PYRKCRwherein X₁ is D-Lys (SEQ ID NO:37).

The skilled person will appreciate that the tertiary structure of thetargeting peptides may depend on the presence of disulfide bridgesbetween the cysteine residues found in the primary structure. In apreferred embodiment, the T22 peptide contains at least one disulfidebridge between cysteines at positions 4 and 17. In another preferredembodiment, the T22 peptide contains at least one disulfide bridgebetween cysteines at positions 8 and 13. In a still more preferredembodiment, the T22 peptide contains a first disulfide bridge betweencysteines at positions 4 and 17 and a second disulfide bridge betweencysteines at positions 8 and 13.

The T22 peptide may further comprise a methionine residue at theN-terminal position.

Suitable functional variants of the targeting peptide are those showinga degree of identity with respect to the T22 peptide of about greaterthan 25% amino acid sequence identity, such as 25% 40%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree ofidentity between two polypeptides is determined using computeralgorithms and methods that are widely known for the persons skilled inthe art. The identity between two amino acid sequences is preferablydetermined by using the BLASTP algorithm as described previously [BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894,Altschul, S., et al., J. Mol. Biol. 1990; 215: 403-410]. The proteins ofthe invention may include post-translational modifications, such asglycosylation, acetylation, isoprenylation, myristoylation, proteolyticprocessing, etc.

Alternatively, suitable functional variants of the targeting peptide arethose wherein one or more positions contain an amino acid which is aconservative substitution of the amino acid present in the T22 proteinmentioned above. “Conservative amino acid substitutions” result fromreplacing one amino acid with another having similar structural and/orchemical properties For example, the following six groups each containamino acids that are conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Selection of suchconservative amino acid substitutions is within the skill of one ofordinary skill in the art and is described, for example by Dordo et al.et al., (J. Mol. Biol, 1999, 217; 721-739) and Taylor et al., (J. Theor.Biol., 1986, 119:205-218).

The first component is capable of specifically binding to CXCR4 andpromoting internalization of the second component.

The term “CXCR4”, as used herein, refers to a G protein-coupled,seven-transmembrane chemokine receptor. Like other chemokine receptors,CXCR4 plays an important role in immune and inflammatory responses bymediating the directional migration and activation of leukocytes CXCR4is expressed or overexpressed in a variety of cancer cell lines andtissues including breast, prostate, lung, ovarian, colon, pancreatic,kidney, and brain, as well as non-Hodgkin's lymphoma and chroniclymphocytic leukemia. The only known ligand to CXCR4 is stromalcell-derived factor-1 (SDF-1, or CXCL12). The interaction between CXCR4and SDF-1 plays an important role in multiple phases of tumorigenesis,including tumor growth, invasion, angiogenesis, and metastasis.

Binding affinity is measured, for instance, as described by Tamamura etal. by the oil-cushion method (see Hesselgesset et al, 1998, J.Immunol., 160:877-883) comprising contacting the peptide withCXCR4-transfected cell line (e.g. CHO cells) and a labeled CXCR4 ligand(e.g. ¹²⁵I-SDF-1α) and measuring the inhibition percentage of thetargeting peptide against the binding of the labeled CXCR4 ligand.

The expression “specifically binding to CXCR4”, as used herein refers tothe ability of the conjugates of the invention to bind more frequently,more rapidly, with greater duration and/or with greater affinity toCXCR4 or cell expressing same than it does with alternative receptors orcells without substantially binding to other molecules.

Binding affinity is measured, for instance, as described by Tamamura etal. by the oil-cushion method (see Hesselgesset et al, 1998, J.Immunol., 160:877-883) comprising contacting the peptide withCXCR4-transfected cell line (e.g. CHO cells) and a labeled CXCR4 ligand(e.g. ¹²⁵I-SDF-1α) and measuring the inhibition percentage of thetargeting peptide against the binding of the labeled CXCR4 ligand.

Specific binding can be exhibited, e.g., by a low affinity targetingagent having a Kd of at least about 10⁻⁴ M. For example, if CXCR4 hasmore than one binding site for a ligand, a ligand having low affinitycan be useful for targeting. Specific binding also can be exhibited by ahigh affinity ligands, e.g. a ligand having a Kd of at least about of10⁻⁷ M, at least about 10⁻⁸ M, at least about 10⁻⁹ M, at least about10⁻¹⁰ M, or can have a Kd of at least about 10⁻¹¹ M or 10⁻¹² M orgreater. Both low and high affinity-targeting ligands are useful forincorporation in the conjugates of the present invention.

As used herein, “internalization” refers to a process by which amolecule or a construct comprising a molecule binds to a target elementon the outer surface of the cell membrane and the resulting complex isinternalized by the cell. Internalization may be followed up bydissociation of the resulting complex within the cytoplasm. The targetelement, along with the molecule or the construct, may then localize toa specific cellular compartment.

The ability of the conjugate of the invention to be internalized bycells expressing CXCR4 may be conveniently determined by fluorescencemethods in the case that the conjugate comprises a fluorescent protein,such as GFP. Such fusion proteins can be obtained by preparing arecombinant nucleic acid wherein the nucleic acids encoding the T22peptide and the fluorescent protein are fused in frame and expressed inan adequate host cell or organism. The fusion protein is then contactedwith a culture of cells expressing CXCR4 or in vivo with a tissue whichexpresses CXCR4 for an appropriate amount of time, after whichfluorescence microscopy may be used to determine whether the constructpenetrated the cell. Presence of fluorescence in the cytoplasm may befurther investigated by comparing the fluorescence microscopy imageresulting from the fluorescent protein to that obtained with a knowncytoplasmic stain.

Alternatively, it is also possible to test the ability of the conjugateof the invention to be internalized by cells expressing CXCR4 bypreparing non-covalent complexes between a fusion protein comprising thefirst component of the conjugate and a polynucleotide encoding afluorescent protein (e.g. the TdTomato gene, encoding a red fluorescentprotein). The complex is then contacted with a culture of cellsexpressing CXCR4 or in vivo with a tissue which expresses CXCR4 for anappropriate amount of time, after which fluorescence microscopy may beused to determine whether the construct penetrated the cell and the geneencoding the fluorescent protein has been expressed. Presence offluorescence in the nucleus may be further investigated by comparing thefluorescence microscopy image resulting from the fluorescent protein tothat obtained with a known nuclear stain (e.g. DAPI).

In a preferred embodiment, the first component is capable ofspecifically binding to CXCR4 and promoting internalization andendosomal escape of the second component.

The expression “promoting endosomal escape”, as used herein, refers tothe ability of the targeting peptide to induce the release of theconjugates from the endosomal compartment after internalization byreceptor-mediated endocytosis.

B. The Therapeutic Agent

The therapeutic agent (also known herein as second component of theconjugate of the invention) is a compound of therapeutic interest.

The term “therapeutic” is used in a generic sense and includes treatingagents, prophylactic agents, and replacement agents.

The nature of the therapeutic agent is not particularly limiting as longas it can be conjugated to the targeting peptide or provided as acomplex with the targeting peptide. Thus, the therapeutic agent may be apolypeptide or a nucleic acid. Thus, the following alternatives oftherapeutic agent are possible:

-   -   The therapeutic agent is a polypeptide which is associated to        the targeting peptide by a covalent bond.    -   The therapeutic agent is a polypeptide and forms a fusion        protein with the targeting peptide.    -   The therapeutic agent is a polynucleotide which may be directly        attached to the targeting peptide or by an additional protein        domain which shows affinity for DNA, particularly by        electrostatic interaction. Suitable protein domains which are        capable of associating with DNA via electrostatic interaction        include, without limitation, polylysine, protamine, albumin and        cationized albumin.    -   The therapeutic agent is a small organic molecule.    -   The therapeutic agent is any of the above (polypeptide,        polynucleotide or small organic molecule) which is provided        within a nanotransporter which is coupled to the targeting        peptide.

In a preferred embodiment, the therapeutic agent is a high molecularweight compound. As used herein, “high molecular weight compound” refersto any chemical entity or molecule, such as nucleic acids, peptides,proteins, natural and synthetic polymers, drugs such as antibiotics andthe like, having a molecular weight greater than 1 kDa, preferablygreater than 5 kDa, more preferably greater than 10 kDa and even morepreferably greater than 100 kDa.

B.1. Polypeptides as Therapeutic Agents

In a preferred embodiment, the therapeutic agent is a polypeptide.

The term “polypeptide”, as used herein, refers to a polymer of aminoacid residues. The term also apply to amino acid polymers in which oneor more amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymer.

In the case that the therapeutic agent is a polypeptide, the conjugatemay be a covalent complex of the therapeutic agent and the targetingpeptide. In another embodiment, the therapeutic agent and the targetingpeptide form a fusion protein. Preferably, wherein the therapeutic agentis a polypeptide and the first and second component form a fusionprotein, then the conjugate is not polyphemusin-1 or polyphemusin-2.

In a preferred embodiment, the polypeptide which acts as active agent orthe fusion protein formed by the said polypeptide and the targetingpeptide further comprises a tag which may be used for the detection orfor the purification of the conjugate using reagents showing specificaffinity towards said tags. Adequate detection/purification tagsincludes hexa-his (metal chelate moiety), hexa-hat GST (glutathioneS-tranferase) glutathione affinity, calmodulin-binding peptide (CBP),strep-tag, cellulose binding domain, maltose binding protein, S-peptidetag, chitin binding tag, immuno-reactive epitopes, epitope tags, E2tag,HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, GIu-GIuepitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-Cepitope, VSV epitope or any other tag as long as the tag does not affectthe stability of the conjugate or the targeting capabilities.

Suitable polypeptides that can be used as therapeutic agents include anypolypeptide which is capable of promoting a decrease in cellproliferation rates.

B.2. Polynucleotides as Therapeutic Agents

In another embodiment, the therapeutic agent forming part of theconjugates of the invention is a nucleic acid. In cases where thetherapeutic agent is a polynucleotide, this is either directlyassociated to the targeting peptide interactions in those cases whereinthe peptide has a net positive charge which allows the formation ofelectrostatic interactions with the DNA. Alternatively, the targetingpeptide may comprise an additional protein domain present in saidtargeting peptide having a net positive charge and thus, capable offorming electrostatic interactions with the DNA. Suitable proteindomains which are capable of associating with DNA via electrostaticinteraction include, without limitation, polylysine, protamine, albuminand cationized albumin.

The terms “nucleic acid” and “polynucleotide”, as used hereininterchangeably, refer to a polymer composed of nucleotide units(ribonucleotides, deoxyribonucleotides, related naturally occurringstructural variants and synthetic non-naturally occurring analogsthereof or combinations thereof) linked via phosphodiester bonds,related naturally occurring structural variants and syntheticnon-naturally occurring analogs thereof.

The nucleic acids include coding regions and the adequate regulatorysignals for promoting expression in the target cells in those caseswherein a defective gene function is to be reinstated in the cell or asilencing nucleic acid wherein the expression of a target gene is to beinhibited.

Generally, nucleic acids containing a coding region will be operablylinked to appropriate regulatory sequences. Such regulatory sequencewill at least comprise a promoter sequence. As used herein, the term“promoter” refers to a nucleic acid fragment that functions to controlthe transcription of one or more genes, located upstream with respect tothe direction of transcription of the transcription initiation site ofthe gene, and is structurally identified by the presence of a bindingsite for DNA-dependent RNA polymerase, transcription initiation sitesand any other DNA sequences, including, but not limited to transcriptionfactor binding sites, repressor and activator protein binding sites, andany other sequences of nucleotides known to one of skill in the art toact directly or indirectly to regulate the amount of transcription fromthe promoter. A “constitutive” promoter is a promoter that is activeunder most physiological and developmental conditions. An “inducible”promoter is a promoter that is regulated depending on physiological ordevelopmental conditions. A “tissue specific” promoter is only active inspecific types of differentiated cells/tissues. Suitable promoters forexpression of the nucleotide sequence encoding the polypeptide from genetherapy vectors include e.g. cytomegalovirus (CMV) intermediate earlypromoter, viral long terminal repeat promoters (LTRs), such as thosefrom murine moloney leukaemia virus (MMLV) rous sarcoma virus, orHTLV-I, the simian virus 40 (SV 40) early promoter and the herpessimplex virus thymidine kinase promoter.

In those cases wherein the nucleic acid includes a coding region, saidcoding region is selected from the group consisting of a tumorsuppressor gene, a suicide gene or a polynucleotide which is capable ofactivating the immune response towards a tumor.

Suitable genes or cDNAs are those which encode the cytotoxic,proapoptotic or metastasis-suppressor polypeptides mentioned above inrelation to the active agent being a polypeptide.

B.3. Small Organic Molecules as Therapeutic Agents

In general, a “small molecule” refers to a substantially non-peptidic,non-oligomeric organic compound either prepared in the laboratory orfound in nature. Small molecules, as used herein, can refer to compoundsthat are “natural product-like,” however, the term “small molecule” isnot limited to “natural product-like” compounds. Rather, a smallmolecule is typically characterized in that it contains severalcarbon-carbon bonds, and has a molecular weight of less than 1500 g/mol,less than 1250 g/mol, less than 1000 g/mol, less than 750 g/mol, lessthan 500 g/mol, or less than 250 g/mol, although this characterizationis not intended to be limiting for the purposes of the presentinvention. In certain other embodiments, natural-product-like smallmolecules are utilized.

Suitable small molecules that can be incorporated in thenanotransporters forming part of the conjugates according to theinvention include, without limitation, anti-cancer agents,anti-angiogenic agents, pro-apoptotic and antiretroviral agents.

B.4. Nanotransporters as Therapeutic Agents

The term “nanotransporter”, as used herein, relates to a multi-componentcomplex with controlled dimensions, e.g., a diameter or radius on theorder of about 1 to about 1000 nanometers that contains a compound ofinterest.

The nanotransporters according to the invention may include one or moreof the active agents (polynucleotide or polypeotides) mentioned above aswell as any other cytotoxic agent suitable for decreasing cellproliferation, including small molecules.

Preferred nanotransporters for use in the present invention includenanoparticles, viruses, virus-like particles (VLP), nanoparticles,protein cages and the like.

B.4.i. Nanotransporters Based on Nanoparticles

In another embodiment, the nanotransporters are nanoparticles.Nanoparticles suitable for use in the present invention include lipidnanoparticles as well as polymeric nanoparticles.

Polymeric nanoparticles are formed by a polymeric matrix which isattached to the T22 peptide targeting moiety. Non-limiting examples ofbiocompatible polymers that may be useful in the polymericnanoparticules according to the present invention include polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, or polyamines,polyglutamate, dextran, polyanhydrides, polyurethanes,polymethacrylates, polyacrylates or polycyanoacrylates.polydioxanone(PDO), polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerolsebacate), polyglycolide, polylactide, PLGA, polycaprolactone orcombinations thereof.

Alternatively, the nanoparticles of the invention may be lipidnanoparticles such as a liposome or a micelle.

Formation of micelles and liposomes from, for example, vesicle-forminglipids, is known in the art. Vesicle-forming lipids refer to lipids thatspontaneously form lipid bilayers above their gel-to-liquid crystallinephase transition temperature range. Such lipids typically have certainfeatures that permit spontaneous bilayer formation, such as close toidentical cross-section areas of their hydrophobic and hydrophilicportions permitting packing into lamellar phases. Lipids capable ofstable incorporation into lipid bilayers, such as cholesterol and itsvarious analogs, can be incorporated into the lipid bilayer duringbilayer formation. The vesicle-forming lipids are preferably lipidshaving two hydrocarbon chains, typically acyl chains, and a head group,either polar or nonpolar. There are a variety of syntheticvesicle-forming lipids and naturally-occurring vesicle-forming lipids,including the phospholipids, such as phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, andsphingomyelin, where the two-hydrocarbon chains are typically betweenabout 14-22 carbon atoms in length, and either saturated or havingvarying degrees of unsaturation. The above-described lipids andphospholipids whose acyl chains have varying degrees of saturation canbe obtained commercially or prepared according to published methods.Other suitable lipids include phospholipids, sphingolipids, glycolipids,and sterols, such as cholesterol.

The term “liposome” refers to vesicles comprised of one or moreconcentrically ordered lipid bilayers, which encapsulate an aqueousphase. The aqueous phase typically contains the compound to be deliveredto a target site. Upon reaching a target site, the liposome fuses withthe plasma membranes of local tumor cells or tumor blood vessel cells,thereby releasing the compound into the cytosol. Alternatively, theliposome is endocytosed or otherwise taken in by the tumor cells or oftumor blood vessel cells as the content of a transport vesicle (e.g., anendosome or phagosome). Once in the transport vesicle, the liposomeeither degrades or fuses with the membrane of the vesicle and releasesits contents. A variety of methods known to the skilled person areavailable for preparing liposomes, such as Suitable methods include, forexample, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of smallliposome vehicles and ether fusion methods, all of which are well knownin the art.

Polymeric and lipidic nanotransporters can additionally include acoating of an amphiphilic compound that surrounds the polymeric materialforming a shell for the particle or a stealth material that can allowthe particles to evade recognition by immune system components andincrease particle circulation half life.

B.4.ii. Viral Nanotransporters

In one embodiment, the nanotransporter of the invention is a virus. Theskilled person will appreciate that any virus known in the art may beused as nanotransporter in the present invention provided thatsufficient information is available so as to allow the modification ofthe external components by T22 peptide or the functionally equivalentvariant thereof. Thus, in the case of non-enveloped viruses, thenanotransporters of the invention are obtained by directly modifying atleast one of the capsid proteins, either by chemical coupling of the T22peptide or the functionally equivalent variant thereof or by insertingthe sequence encoding the T22 peptide or the functionally equivalentvariant thereof into the viral gene coding for the capsid protein sothat, upon synthesis and assembly into the capsid, the T22 peptide orthe functionally equivalent variant thereof is exposed to the outersurface of the capsid. Examples of suitable virus capsids that can bemodified in the above manner include, but are not limited to, capsidsfrom Sindbis virus and other alphaviruses, rhabdoviruses (e.g. vesicularstomatitis virus), picornaviruses (e.g., human rhino virus, Aichivirus), togaviruses (e.g., rubella virus), orthomyxoviruses (e.g.,Thogoto virus, Batken virus, fowl plague virus), polyomaviruses (e.g.,polyomavirus BK, polyomavirus JC, avian polyomavirus BFDV),parvoviruses, rotaviruses, bacteriophage Qβ, bacteriophage P1,bacteriophage M13, bacteriophage MS2, bacteriophage G4, bacteriophageP2, bacteriophage P4, bacteriophage 186, bacteriophage Φ6, bacteriophageΦ29, bacteriophage MS2, bacteriophage N4, bacteriophage ΦX174,bacteriophage AP205, Norwalk virus, foot and mouth disease virus, aretrovirus, Hepatitis B virus, Tobacco mosaic virus (TMV), satellitepanicum mosaic virus (SPMV), flock house virus and human papilomavirus.

Alternatively, wherein the nanotransporter is an enveloped virus, thetargeting peptide is preferably attached to or replacing a part of theenvelope glycoproteins. Some non-limiting examples of surfaceglycoproteins that may be used for inserting the short fiber proteininclude glycoproteins from alphaviruses, such as Semliki Forest virus(SFV), Ross River virus (RRV) and Aura virus (AV), which comprisesurface glycoproteins such as E1, E2, and E3. The E2 glycoproteinsderived from the Sindbis virus (SIN) and the hemagglutinin (HA) ofinfluenza virus are non-retroviral glycoproteins that specifically bindparticular molecules on cell surfaces (heparin sulfate glycosaminoglycanfor E2, sialic acid for HA) which are known to tolerate certain geneticmodifications and remain efficiently assembled on the retroviral surface(Morizono et al. J. Virol. 75, 8016-8020); glycoproteins of Lassa fevervirus, Hepatitis B virus, Rabies virus, Borna disease virus, Hantaanvirus, or SARS-CoV; flavivirus-based surface glycoproteins,hemagglutinin (HA) from influenza A/fowl plague virus/Rostock/34 (FPV),a class I fusogen, is used (T. Hatziioannou, S. Valsesia-Wittmann, S. J.Russell, F. L. Cosset, J. Virol. 72, 5313 (1998)). Suitables viruses foruse in the present invention comprise alphaviruses, paramyxoviruses,rhabdoviruses, coronaviruses, picornaviruses, myxoviruses, reoviruses,bunyaviruses, flaviviruses, rubiviruses, filoviruses, arenaviruses,arteriviruses or caliciviruses.

B. 4.iii. Nanotransporters Based on VLPs

In one embodiment, the nanotransporter used in the present invention isa VLP. The term “VLP or viral-like particle”, as used herein, relates toa self-assembling macromolecular structure formed by the viralnucleocapsids which acquire a quaternary structure resembling that ofthe virus from which they originate but which are devoid of the virusgenetic material.

The VLPs for use according to the present invention may be formed frompolypeptides derived from any virus known in the art having an orderedand repetitive structure. VLPs can be produced and purified fromvirus-infected cell culture. The resulting virus or virus-like particlefor vaccine purpose needs to be devoid of virulence. Besides geneticengineering, physical or chemical methods can be employed to inactivatethe viral genome function, such as UV irradiation, formaldehydetreatment. Alternatively, the VLP is a recombinant VLP. The skilledperson will appreciate that almost all commonly known viruses of knownsequence may be used for the generation of VLPs provided that the geneencoding the coat protein or proteins can be easily identified by askilled artisan. The preparation of VLPs by the recombinant expressionof the coat protein or proteins in a host is within the common knowledgeof a skilled artisan. Suitable VLPS can be obtained from thenucleocapsid proteins of a virus selected form the group consisting ofRNA-bacteriophages, adenovirus, papaya mosaic virus, influenza virus,norovirus, papillomavirus, hepadnaviridae, respiratory syncytial virus,hepatitis B virus, hepatitis C virus, measles virus; Sindbis virus;rotavirus, foot-and-mouth-disease virus, Newcastle disease virus,Norwalk virus, alphavirus; SARS, paramoxyvirus, transmissiblegastroenteritis virus retrovirus, retrotransposon Ty, Polyoma virus;tobacco mosaic virus; Flock House Virus, Cowpea Chlorotic Mottle Virus;a Cowpea Mosaic Virus; and alfalfa mosaic virus.

B.4.iv. Nanotransporters Based on Protein Cages

In another embodiment, the nanotransporter used in the present inventionis a protein cage. The term “protein cage”, as used herein, relates toself-assembling macromolecular structure formed by one or more differentproteins which are capable of forming a constrained internalenvironment. Protein cages can have different core sizes, ranging from 1to 30 nm (e.g., the internal diameter of the shells). Preferred proteincages suitable for use in the present invention include ferritin proteincages, heat-shock protein cages as described in WO08124483 and the like.

C. Linker Regions

The conjugates object of the invention comprising the targeting peptideand the therapeutic agent wherein said therapeutic agent has a peptidicnature can contain a bond directly connecting the targeting peptide andthe therapeutic agent or, alternatively, can contain an additional aminoacid sequence acting as a linker between the targeting peptide and thetherapeutic agent. The linker peptide preferably comprises at least twoamino acids, at least three amino acids, at least five amino acids, atleast ten amino acids, at least 15 amino acids, at least 20 amino acids,at least 30 amino acids, at least 40 amino acids, at least 50 aminoacids, at least 60 amino acids, at least 70 amino acids, at least 80amino acids, at least 90 amino acids or approximately 100 amino acids.

According to the invention, said non-natural intermediate amino acidsequence acts as a hinge region between domains, allowing them to moveindependently from one another while they maintain the three-dimensionalshape of the individual domains. In this sense, a preferred non-naturalintermediate amino acid sequence according to the invention would be ahinge region characterized by a structural ductility allowing thismovement. In a particular embodiment, said non-natural intermediateamino acid sequence is a non-natural flexible linker. In a preferredembodiment, said flexible linker is a flexible linker peptide with alength of 20 amino acids or less. In a more preferred embodiment, thelinker peptide comprises 2 amino acids or more selected from the groupconsisting of glycine, serine, alanine and threonine. In a preferredembodiment of the invention, said flexible linker is a polyglycinelinker.

Preferred examples of spacer or linker peptides include those which havebeen used for binding proteins without substantially deteriorating thefunction of the bound proteins or at least without substantiallydeteriorating the function of one of the bound proteins. Morepreferably, the spacers or linkers have been used for binding proteinscomprising structures with coiled helixes such as:

-   -   the peptide GTKVHMK (SEQ ID NO:18) formed by residues 53-56 and        residues 57-59 of tetranectin (Nielsen, B.B. et al., FEBS Lett.        412: 388-396, 1997);    -   the connecting strand 3 from human fibronectin, corresponding to        amino acids 1992-2102 (SWISSPROT numbering, entry P02751).    -   The subsequence PGTSGQQPSVGQQ (SEQ ID NO: 19) corresponding to        amino acids number 2037-2049 of fibronectin and within that        subsequence fragment GTSGQ (SEQ ID NO: 20) corresponding to        amino acids 2038-2042.    -   The 10 amino acid residue sequence of the upper hinge region of        murine IgG3 (PKPSTPPGSS, SEQ ID NO: 21). In a preferred        embodiment, the linker peptide is selected from the group of the        peptide of sequence APAETKAEPMT (SEQ ID NO: 22) and of the        peptide of sequence GAP.    -   The eight amino acid sequence GGSSRSSS (SEQ ID NO:32).

Alternatively, the two components of the conjugates of the invention canbe connected by a peptide the sequence of which contains a cleavagetarget for a protease, thus allowing the separation of the targetingpeptide from the therapeutic agent. Protease cleavage sites suitable fortheir incorporation into the polypeptides of the invention includeenterokinase (cleavage site DDDDK, SEQ ID NO: 23), factor Xa (cleavagesite IEDGR, SEQ ID NO: 24), thrombin (cleavage site LVPRGS, SEQ ID NO:25), TEV protease (cleavage site ENLYFQG, SEQ ID NO: 26), PreScissionprotease (cleavage site LEVLFQGP, SEQ ID NO: 27), inteins and the like.

D. Polynucleotides, Vectors and Host Cells

In those cases wherein the conjugate is a fusion protein of thetargeting peptide and the active (therapeutic or diagnostic) agent, thenthe fusion protein can be produced in vivo by the recipient subject whena polynucleotide encoding the fusion protein is used. Thus, in anotheraspect, the invention relates to a polynucleotide encoding a conjugateaccording to the invention.

The expressions “nucleotide sequence”, “nucleic acid” and“polynucleotide” are used interchangeably in this invention to refer tothe polymer form of phosphate esters of ribonucleosides (adenosine,guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides(deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; “DNAmolecules”), or any analogous phosphoester thereof, such asphosphorothioates and thioesters, in single-strand or double-strandform. Thus, helices formed by DNA-DNA, DNA-RNA and RNA-RNA are possible.The term “nucleic acid sequence” and, in particular, DNA or RNAmolecule, refers solely to the primary or secondary structure of themolecule and does not limit any particular type of tertiary structure.Thus, this term includes double-chain DNA as it appears in linear orcircular DNA molecules, supercoiled DNA plasmids and chromosomes.

In another aspect, the invention relates to a vector comprising apolynucleotide according to the invention.

As used in this invention, the term “vector” refers to a vehicle wherebya polynucleotide or a DNA molecule may be manipulated or introduced intoa cell. The vector may be a linear or circular polynucleotide, or it maybe a larger-size polynucleotide or any other type of construct, such asDNA or RNA from a viral genome, a virion or any other biologicalconstruct that allows for the manipulation of DNA or the introductionthereof into the cell. It is understood that the expressions“recombinant vector” and “recombinant system” may be usedinterchangeably with the term “vector”. Those skilled in the art willnote that there is no limitation in terms of the type of vector that maybe used, since said vector may be a cloning vector suitable forpropagation and to obtain the adequate polynucleotides or geneconstructs or expression vectors in different heterologous organismssuitable for the purification of the conjugates. Thus, suitable vectorsin accordance with this invention include expression vectors inprokaryotes, such as pUC18, pUC19, Bluescript and the derivativesthereof, mp18, mp19, pBR322, pMB9, CoIE1, pCR1, RP4, phages and“shuttle” vectors, such as pSA3 and pAT28, expression vectors in yeasts,such as vectors of the 2-micron plasmid type, integration plasmids, YEPvectors, centromere plasmids and similar ones, expression vectors ininsect cells, such as the vectors in the pAC series and the pVL series,expression vectors in plants, such as vectors from the pIBI,pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series andsimilar ones, and expression vectors in higher eukaryotic cells based onviral vectors (adenoviruses, viruses associated with adenoviruses, aswell as retroviruses and lentiviruses) and non-viral vectors, such aspSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg, pHCMV/Zeo, pCR3.1,pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His,pVAX1, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDT1.

The vector of the invention may be used to transform, transfect orinfect cells susceptible to being tranformed, transfected or infected bysaid vector.

Therefore, another aspect of the invention relates to a cell thatcomprises a polynucleotide, a gene construct or a vector of theinvention; to this end, said cell has been transformed, transfected orinfected with a construct or vector provided by this invention.Transformed, transfected or infected cells may be obtained byconventional methods known to those skilled in the art (Sambrook et al.,2001, cited supra). In a particular embodiment, said host cell is ananimal cell transfected or infected with an appropriate vector.

Host cells suitable for the expression of the conjugates of theinvention include, without being limited thereto, cells from mammals,plants, insects, fungi and bacteria. Bacterial cells include, withoutbeing limited thereto, cells from Gram-positive bacteria, such asspecies from the genera Bacillus, Streptomyces and Staphylococcus, andcells from Gram-negative bacteria, such as cells from the generaEscherichia and Pseudomonas. Fungi cells preferably include cells fromyeasts such as Saccharomyces, Pichia pastoris and Hansenula polymorpha.Insect cells include, without limitation, Drosophila cells and Sf9cells. Plant cells include, amongst others, cells from cultivatedplants, such as cereals, medicinal plants, ornamental plants or bulbs.Mammalian cells suitable for this invention include epithelial celllines (porcine, etc.), osteosarcoma cell lines (human, etc.),neuroblastoma cell lines (human, etc.), epithelial carcinomas (human,etc.), glial cells (murine, etc.), hepatic cell lines (from monkeys,etc.), CHO (Chinese Hamster Ovary) cells, COS cells, BHK cells, HeLa,911, AT1080, A549, 293 or PER.C6 cells, human NTERA-2 ECC cells, D3cells from the mESC line, human embryonary stem cells, such as HS293 andBGV01, SHEF1, SHEF2 and HS181, NIH3T3, 293T, REH and MCF-7 cells, andhMSC cells.

E. Conjugates of the Invention Comprising Antitumor Agents and UsesThereof for the Treatment of Cancer

The efficient and specific interaction of the conjugates of theinvention with CXCR4+ cells allows the use of said conjugates for thetreatment of any disease wherein it is desirable to deliver a compoundof interest to cells expressing CXCR4. Thus, in another aspect, theinvention relates to a conjugate, a polynucleotide, or a vectoraccording to the invention wherein the therapeutic agent is an antitumoragent.

The term “antitumor agent”, as used herein, refers to any chemical orbiological agent or compound with antiproliferative, antioncogenicand/or carcinostatic properties which can be used to inhibit tumorgrowth, proliferation and/or development

Suitable antitumor agents for use in the present invention include,without limitation:

-   -   (i) a cytotoxic polypeptide,    -   (ii) an antiangiogenic polypeptide,    -   (iii) a polypeptide encoded by a tumor suppressor gene,    -   (iv) a polypeptide encoded by a polynucleotide which is capable        of activating the immune response towards a tumor,    -   (v) a tumor suppressor gene,    -   (vi) a silencing agent,    -   (vii) a suicide gene,    -   (viii) a polynucleotide which is capable of activating the        immune response towards a tumor,    -   (ix) a chemotherapy agent and    -   (x) an antiangiogenic molecule.

E.1. Cytotoxic Polypeptides

As used herein, the term cytotoxic polypeptide refers to an agent thatis capable of inhibiting cell function. The agent may inhibitproliferation or may be toxic to cells. Any polypeptide that wheninternalized by a cell interfere with or detrimentally alter cellularmetabolism or in any manner inhibit cell growth or proliferation areincluded within the ambit of this term, including, but are not limitedto, agents whose toxic effects are mediated when transported into thecell and also those whose toxic effects are mediated at the cellsurface. Useful cytoxic polypeptides include proteinaceous toxins andbacterial toxins.

Examples of proteinaceous cell toxins useful for incorporation into theconjugates according to the invention include, but are not limited to,type one and type two ribosome inactivating proteins (RIP). Useful typeone plant RIPs include, but are not limited to, dianthin 30, dianthin32, lychnin, saporins 1-9, pokeweed activated protein (PAP), PAP II,PAP-R, PAP-S, PAP-C, mapalmin, dodecandrin, bryodin-L, bryodin, Colicin1 and 2, luffin-A, luffin-B, luffin-S, 19K-protein synthesis inhibitoryprotein (PSI), 15K-PSI, 9K-PSI, alpha-kirilowin, beta-kirilowin,gelonin, momordin, momordin-II, momordin-Ic, MAP-30, alpha-momorcharin,beta-momorcharin, trichosanthin, TAP-29, trichokirin; barley RIP; flaxRIP, tritin, corn RIP, Asparin 1 and 2 (Stirpe et al., Bio/Technology10:405-12, 1992). Useful type two RIPs include, but are not limited to,volkensin, ricin, nigrin-b, CIP-29, abrin, modeccin, ebulitin-[beta],ebultin-[gamma], vircumin, porrectin, as well as the biologically activeenzymatic subunits thereof (Stirpe et al., Bio/Technology 10:405-12,1992; Pastan et al., Annu. Rev. Biochem. 61:331-54; Brinkmann andPastan, Biochim. et Biophys. Acta 1198:27-45, 1994; and Sandvig and VanDeurs, Physiol. Rev. 76:949-66, 1996).

Examples of bacterial toxins useful as cell toxins include, but are notlimited to, shiga toxin and shiga-like toxins (i.e., toxins that havethe same activity or structure), as well as the catalytic subunits andbiologically functional fragments thereof. These bacterial toxins arealso type two RIPs (Sandvig and Van Deurs, Physiol. Rev. 76:949-66,1996; Armstrong, J. Infect. Dis., 171:1042-5, 1995; Kim et al.,Microbiol. Immunol. 41:805-8, 1997, and Skinner et al., Microb. Pathog.24:117-22, 1998). Additional examples of useful bacterial toxinsinclude, but are not limited to, Pseudomonas exotoxin and Diphtheriatoxin (Pastan et al., Annu. Rev. Biochem. 61:331-54; and Brinkmann andPastan, Biochim. et Biophys. Acta 1198:27-45, 1994). Truncated forms andmutants of the toxin enzymatic subunits also can be used as a cell toxinmoiety (Pastan et al., Annu. Rev. Biochem. 61:331-54; Brinkmann andPastan, Biochim. et Biophys. Acta 1198:27-45, 1994; Mesri et al., J.Biol. Chem. 268:4852-62, 1993; Skinner et al., Microb. Pathog.24:117-22, 1998; and U.S. Pat. No. 5,082,927). Other targeted agentsinclude, but are not limited to the more then 34 described Colicinfamily of RNase toxins which include colicins A, B, D, E1-9, cloacinDF13 and the fungal RNase, [alpha]-sarcin (Ogawa et al. Science 283:2097-100, 1999; Smarda et al., Folia Microbiol (Praha) 43:563-82, 1998;Wool et al., Trends Biochem. Sci., 17: 266-69, 1992).

E. 2. Antiangiogenic Peptides and Polypeptides

Proliferation of tumors cells relies heavily on extensive tumorvascularization, which accompanies cancer progression. Thus, inhibitionof new blood vessel formation with anti-angiogenic agents and targeteddestruction of existing blood vessels have been introduced as aneffective and relatively non-toxic approach to tumor treatment.

The term “anti-angiogenic polypeptide”, as used herein, denotes apolypeptide capable of inhibiting angiogenesis. Suitable antiangiogenicpolypeptides include, without limitation, angiostatin, endostatin,anti-angiogenic anti-thrombin III, sFRP-4 as described in WO2007115376,an anti-VEGF antibody such as anibizumab, bevacizumab (avastin), Fab IMC1121 and F200 Fab.

E. 3. Polypeptides Encoded by Tumor Suppressor Genes,

As used herein, a “tumor suppressor” is a gene or gene product that hasa normal biological role of restraining unregulated growth of a cell.The functional counterpart to a tumor suppressor is an oncogene. Genesthat promote normal cell growth may be known as “protooncogenes”. Amutation that activates such a gene or gene product further converts itto an “oncogene”, which continues the cell growth activity, but in adysregulated manner. Examples of tumor suppressor genes and geneproducts are well known in the literature and may include PTC, BRCA1,BRCA2, p16, APC, RB, WT1, EXT1, p53, NF1, TSC2, NF2, VHL,ST7, ST14,PTEN, APC, CD95 or SPARC.

E.4. Pro-Apoptotic Polypeptides

The term “pro-apoptotic polypeptides”, as used herein, refers to aprotein which is capable of inducing cell death in a cell or cellpopulation. Suitable pro-apoptotic polypeptides include, withoutlimitation, proapoptotic members of the BCL-2 family of proteins such asBAX, BAK, BOK/MTD, BID, BAD, BIK/NBK, BLK, HRK, BIM/BOD, BNIP3, NIX,NOXA, PUMA, BMF, EGL-I, and viral homologs, caspases sich as caspase-8,the adenovirus E4orf4 gene, p53 pathway genes, proapoptotic ligands suchas TNF, FasL, TRAIL and/or their receptors, such as TNFR, Fas, TRAIL-R1and TRAIL-R2.

E.5. Polypeptides Having Anti-Metastatic Activity

The term “metastasis suppressor” as used herein, refers to a proteinthat acts to slow or prevent metastases (secondary tumors) fromspreading in the body of an organism with cancer. Suitable metastasissuppressors include, without limitation, proteins such as BRMS 1, CRSP3,DRG1, KAI1, KISS-1, NM23, a TIMP-family protein and uteroglobin.

E. 6. Immunostimulatory Polypeptides

As used herein, an immunostimulatory polypeptide agent is a polypeptidethat stimulates an immune response (including enhancing a pre-existingimmune response) in a subject to whom it is administered, whether aloneor in combination with another agent, flagellin, muramyl dipeptide),cytokines including interleukins (e.g., IL-2, IL-7, IL-15 (orsuperagonist/mutant forms of these cytokines), IL-12, IFN-gamma,IFN-alpha, GM-CSF, FLT3-ligand, etc.), immunostimulatory antibodies(e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain/antibodyfragments of these molecules), and the like.

E. 7. Tumor Suppressor Genes

Suitable tumor suppressor genes include a gene or gene product thatencodes any of the polypeptides defined above. Examples of tumorsuppressor genes and gene products are well known in the literature andmay include PTC, BRCA1, BRCA2, p16, APC, RB, WT1, EXT1, p53, NF1, TSC2,NF2, VHL,ST7, ST14, PTEN, APC, CD95 or SPARC.

E. 8. Silencing Agents

Wherein the nucleic acid is a silencing agent, said silencing agent isaimed at blocking the expression of genes the over-expression of whichleads to cell proliferation and tumor growth. Genes that can beinhibited by the conjugates according to the invention carryingsilencing agent include, without limitation, HRAS (v-Ha-ras Harvey ratsarcoma viral oncogene homolog), NRAS (neuroblastoma RAS viral (v-ras)oncogene homolog), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog), MYC (v-myc myelocytomatosis viral oncogene homolog, avian),MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastomaderived, avian), MYB (v-myb myeloblastosis viral oncogene homolog,avian), Jun-oncogene, FOS (v-fos FBJ murine osteosarcoma viral oncogenehomolog), Oncogenic ABL1 (v-abl Abelson murine leukemia viral oncogenehomolog 1 (this gene is an oncogene only if the SH3 domain is truncated,as happens regularity in certain leukemias), SRC (v-src sarcoma(Schmidt-Ruppin A-2) viral oncogene homolog, avian), FES (Feline sarcomaoncogene), RAF1(v-raf-1 murine leukemia viral oncogene homolog 1), REL(v-rel reticuloendotheliosis viral oncogene homolog, avian), RELA (v-relreticuloendotheliosis viral oncogene homolog A, nuclear factor of kappalight polypeptide gene enhancer in B-cells 3, p65, avian), RELB (v-relreticuloendotheliosis viral oncogene homolog B, nuclear factor of kappalight polypeptide gene enhancer in B-cells 3, avian), FGR(Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog), and KIT(v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog).

Other silencing agents suitable for use in the present invention includethose directed against cyclin G1, cyclin D1, Bc1-2 (B-cell chroniclymphatic leukemia associated protein 2), Bcl-XL (Bcl-2 related genexl), HLA-G (Human leukocyte antigen class G), IGF-1 (Insulin-like growthfactor-1), EGF (epidermal growth factor), FGF (fibroblast growthfactor), VEGF (vascular endothelial growth factor), VEGFR (vascularendothelial growth factor receptor), IGFR (insulin-like growth factorreceptor), EGFR (epidermal growth factor receptor), FGFR (fibroblastgrowth factor receptor), TGF-beta (transforming growth factor beta),Caspase 3, CEACAM6 (Carcinoembryonic antigen-related cell adhesionmolecule 6), HPV-E6 (human papiloma virus protein E6), HPV-E7 (humanpapiloma virus protein E7), H-Ras gene, P100a gene, CREB (cAMP responseelement binding), BRAF gene, ATF2 (activating transcription factor 2),HER2 (Human EGF-like Receptor No. 2), and N-myc.

Examples of therapeutic molecules include, but are not limited to, cellcycle control agents; agents which inhibit cyclin proteins, such asantisense polynucleotides to the cyclin G1 and cyclin D1 genes.

In another embodiment, the silencing agent can be targeted againstCXCR4. Suitable CXCR4-silencing agents include, without limitation:

-   -   The antisense oligonucleotides to CXCR4 having the sequences        5′-CTGATCCCCTCCATGGTAACCGCT-3′ (SEQ ID NO: 10),        5′-TATATACTGATCCCCTCCATGGTA-3′ (SEQ ID NO: 11) and        5-CCTCCATGGTAACCGCTGGTTCT-3′ (SEQ ID NO: 12) and described in        U.S. Pat. No. 6,429,308;    -   The CXCR4-specific siRNAs as described in WO2008008852        comprising sense strands having the sequences        5′-UAAAAUCUUCCUGCCCACCdTdT-3′ (SEQ ID NO: 13) and        5′-GGAAGCUGUUGGCUGAAAAdTdT-3′ (SEQ ID NO: 14).    -   The CXCR4-specific interference RNAs targeting the sequences        within CXCR4 mRNA described in WO2007143584.    -   The CXCR4-specific interference RNAs targeting the sequences        within CXCR4 mRNA described in US20050124569.    -   The CXCR4-specific ribozyme having the sequence        3′-UGUUGCA-X-Y-X-UCACUC-5′ wherein X are the catalytic sequences        and Y is the sequence of a stem-loop region. Detailed structures        of this ribozyme and the DNA-cassetes encoding said sequences        are described in U.S. Pat. No. 6,916,653;    -   The CXCR4-specific shRNA having the sequence        5′-GATCCAGGATGGTGGTGTTTCAATTCCTTCAAGAGAGGAATTGAAACACCACCA        TCCTTTTTGG-3 (SEQ ID NO: 15) as described in US2009210952.    -   The CXCR4-specific siRNAs targeting the sequences within the        CXCR4 mRNA AATAAAATCTTCCTGCCCACC (SEQ ID NO: 16) and        AAGGAAGCTGTTGGCTGAAAA (SEQ ID NO: 17) as described in        WO2004087068.    -   The CXCR4-specific interference RNAs targeting the sequences        within CXCR4 mRNA described in US2009235772 and US2005124569.

Potential target sites in the mRNA for the design of RNA-interferenceagents are identified based on rational design principles, which includetarget accessibility and secondary structure prediction. Each of thesemay affect the reproducibility and degree of knockdown of expression ofthe mRNA target, and the concentration of siRNA required for therapeuticeffect. In addition, the thermodynamic stability of the siRNA duplex(e.g., antisense siRNA binding energy, internal stability profiles, anddifferential stability of siRNA duplex ends) may be correlated with itsability to produce RNA interference. (Schwarz et al., Cell 115:199-208,2003; Khvorova et al., Cell 115:209-216, 2003). Empirical rules, such asthose provided by the Tuschl laboratory (Elbashir et al., Nature411:494-498, 2001; Elbashir et al., Genes Dev. 15:188-200, 2001) arealso used. Software and internet interactive services for siRNA designare available at the Ambion and Invitrogen websites. Levenkova et al.describe a software system for design and prioritization of siRNA oligos(Levenkova et al., Bioinformatics 20:430-432, 2004). The Levenkovasystem is available on the internet and is downloadable freely for bothacademic and commercial purposes. The siRNA molecules disclosed hereinwere based on the Ambion, Invitrogen and Levenkova recommendations.

Typically, siRNA oligos specific for a given target is carried out basedprimarily on uniqueness vs. human sequences (i.e., a single good hit vshuman Unigene, and a big difference in hybridization temperature Tmagainst the second best hit) and on GC content (i.e., sequences withpercent GC in the range of 40-60 percent). Optionally, for a moredetailed picture on the potential hybridization of the oligos, RNAtarget accessibility and secondary structure prediction can be carriedout using, for example, Sfold software (Ding Y and Lawrence, C. E.(2004) Rational design of siRNAs with Sfold software. In: RNAInterference: from Basic Science to Drug Development. K. Appasani (Ed.),Cambridge University Press; Ding and Lawrence, Nucleic Acids Res.29:1034-1046, 2001; Nucleic Acids Res. 31:7280-7301, 2003). Sfold isavailable on the internet. RNA secondary structure determination is alsodescribed in Current Protocols in Nucleic Acid Chemistry, Beaucage etal., ed, 2000, at 11.2.1-11.2.10.

The type and mode of action of the silencing agent is not particularlylimiting in the context of the present invention. Suitable silencingagents include, without limitation, antisense RNA or DNA, ribozymes andother oligonucleotides that are intended to be used as antisense agents.Suitable silencing agents against a gene of interest can be identifiedusing standard techniques to detect expression levels of a gene, such asRT-PCR, Northern blot and the like.

Antisense oligonucleotides, including, antisense oligonucleotides;triplex molecules; dumbbell oligonucleotides; Extracellular ProteinBinding Oligonucleotides; and Small Nucleotide Molecules, areoligonucleotides that specifically bind to mRNA that has complementarysequences, thereby preventing translation of the mRNA (see, e.g., U.S.Pat. No. 5,168,053 to Altman et al. U.S. Pat. No. 5,190,931 to Inouye,U.S. Pat. No. 5,135,917 to Burch; U.S. Pat. No. 5,087,617 to Smith andClusel et al. (1993) Nucl. Acids Res. 21:3405-3411, which describesdumbbell antisense oligonucleotides). Triplex molecules refer to singleDNA strands that target duplex DNA and thereby prevent transcription(see, e.g., U.S. Pat. No. 5,176,996 to Hogan et al. which describesmethods for making synthetic oligonucleotides that bind to target siteson duplex DNA).

E.9. Suicide Genes

In another embodiment, the polynucleotide used as active agent comprisesthe coding region of a suicide gene. The term “suicide gene” refers to anucleic acid coding for a product, wherein the product causes cell deathby itself or in the presence of other compounds. A representativeexample of a suicide gene is one, which codes for thymidine kinase ofherpes simplex virus. Additional examples are thymidine kinase ofvaricella zoster virus and the bacterial gene cytosine deaminase, whichcan convert 5-fluorocytosine to the highly cytotoxic compound5-fluorouracil.

Suicide genes may produce cytotoxicity by converting a prodrug to aproduct that is cytotoxic. In one embodiment, the term “prodrug” meansany compound that can be converted to a toxic product for cells. Arepresentative example of such a prodrug is gancyclovir which isconverted in vivo to a toxic compound by HSV-thymidine kinase.

The gancyclovir derivative subsequently is toxic to cells. Otherrepresentative examples of prodrugs include acyclovir, FIAU[1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil],6-methoxypurine arabinoside for VZV-TK, and 5-fluorocytosine forcytosine deaminase.

E.10. Polynucleotides Capable of Activating the Immune Response Towardsa Tumor

Suitable polynucleotides capable of activating the immune responsetowards a tumor include a gene that encodes any immunostimulatorypolypeptide agent as defined above.

E.11. Chemotherapy Agents

As used herein, an anti-cancer agent is an agent that at least partiallyinhibits the development or progression of a cancer, includinginhibiting in whole or in part symptoms associated with the cancer evenif only for the short term. Several anti-cancer agents can becategorized as DNA damaging agents and these include topoisomeraseinhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide,mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine,cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan,thiotepa, carmustine, lomustine, carboplatin, dacarbazine,procarbazine), DNA strand break inducing agents (e.g., bleomycin,doxorubicin, daunorubicin, idarubicin, mitomycin C), anti-microtubuleagents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g.,cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine,6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin,chlorodeoxyadenosine), anthracyclines, vinca alkaloids, orepipodophyllotoxins.

Examples of anti-cancer agents include without limitation Acivicin;Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Bortezomib (VELCADE); Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin (aplatinum-containing regimen); Carmustine; Carubicin Hydrochloride;Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin (aplatinum-containing regimen); Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin;Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel(TAXOTERE); Doxorubicin; Droloxifene; Dromostanolone; Duazomycin;Edatrexate; Eflornithine; Elsamitrucin; Enloplatin; Enpromate;Epipropidine; Epirubicin; Erbulozole; Erlotinib (TARCEVA), Esorubicin;Estramustine; Etanidazole; Etoposide; Etoprine; Fadrozole; Fazarabine;Fenretinide; Floxuridine; Fludarabine; 5-Fluorouracil; Flurocitabine;Fosquidone; Fostriecin; Gefitinib (IRES SA), Gemcitabine; Hydroxyurea;Idarubicin; Ifosfamide; Ilmofosine; Imatinib mesylate (GLEEVAC);Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-n1;Interferon alpha-n3; Interferon beta-I a; Interferon gamma-I b;Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLLM1D, REVIMID);Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine; Losoxantrone;Masoprocol; Maytansine; Mechlorethamine; Megestrol; Melengestrol;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin; Mitosper; Mitotane; Mitoxantrone; Mycophenolic Acid;Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed(ALIMTA), Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin;Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate;Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin;Prednimustine; Procarbazine; Puromycin; Pyrazofurin; Riboprine;Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside; Sparfosate;Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin; Streptonigrin;Streptozocin; Sulofenur; Talisomycin; Tamsulosin; Taxol; Taxotere;Tecogalan; Tegafur; Teloxantrone; Temoporfin; Temozolomide (TEMODAR);Teniposide; Teroxirone; Testolactone; Thalidomide (THALOMID) andderivatives thereof; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;Tirapazamine; Topotecan; Toremifene; Trestolone; Triciribine;Trimetrexate; Triptorelin; Tubulozole; Uracil; Mustard; Uredepa;Vapreotide; Verteporfin; Vinblastine; Vincristine; Vindesine;Vinepidine; Vinglycinate; Vinleurosine; Vinorelbine; Vinrosidine;Vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin.

The anti-cancer agent may be an enzyme inhibitor including withoutlimitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinaseinhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may bewithout limitation Genistein (4′,5,7-trihydroxyisoflavone), Tyrphostin25 (3,4,5-trihydroxyphenyl), methylene]-propanedinitrile, Herbimycin A,Daidzein (4′,7-dihydroxyisoflavone), AG-126,trans-1-(3′-carboxy-4′-hydroxyphenyl)-2-(2″,5″-dihydroxy-phenyl)ethane,or HDBA (2-Hydroxy5-(2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid.The CDK inhibitor may be without limitation p21, p27, p57, p15, p16,p18, or p19. The MAP kinase inhibitor may be without limitation KY12420(C₂₃H₂₄O₈), CNI-1493, PD98059, or 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole. The EGFR inhibitor may be withoutlimitation erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazolinederivative), LFM-Al2 (leflunomide metabolite analog), ABX-EGF,lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.

The anti-cancer agent may be a VEGF inhibitor including withoutlimitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib(MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA),anecortave (RETAANE), squalamine lactate, and semaphorin. Theanti-cancer agent may be an antibody or an antibody fragment includingwithout limitation an antibody or an antibody fragment including but notlimited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab(CAMPATH, indicated for B cell chronic lymphocytic leukemia,),gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such asacute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR,anti-CD20, indicated for B cell malignancy), MDX-210 (bispecificantibody that binds simultaneously to HER-2/neu oncogene protein productand type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)),oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab(PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated forrespiratory conditions such as RSV infection), ibritumomab tiuxetan(ZEVALIN, indicated for Non-Hodgkin's lymphoma), cetuximab (ERBITUX),MDX-447, MDX-22, MDX-220 (anti-TAG-72), I0R-C5, 10R-T6 (anti-CD 1), IOREGF/R3, celogovab (ONCOSCINT OV 103), epratuzumab (LYMPHOCIDE),pemtumomab (THERAGYN), and Gliomab-H (indicated for brain cancer,melanoma).

Other suitable active agents are DNA cleaving agents. Examples of DNAcleaving agents suitable for inclusion as the cell toxin in theconjugates used in practicing the methods include, but are not limitedto, anthraquinone-oligopyrrol-carboxamide, benzimidazole, leinamycin;dynemycin A; enediyne; as well as biologically active analogs orderivatives thereof (i.e., those having a substantially equivalentbiological activity). Known analogs and derivatives are disclosed, forexamples in Islam et al., J. Med. Chem. 34 2954-61, 1991; Skibo et al.,J. Med. Chem. 37:78-92, 1994; Behroozi et al., Biochemistry 35:1568-74,1996; Helissey et al., Anticancer Drug Res. 11:527-51, 1996; Unno etal., Chem. Pharm. Bull. 45:125-33, 1997; Unno et al., Bioorg. Med.Chem., 5:903-19, 1997; Unno et al., Bioorg. Med. Chem., 5: 883-901,1997; and Xu et al., Biochemistry 37:1890-7, 1998). Other examplesinclude, but are not limited to, endiyne quinone imines (U.S. Pat. No.5,622,958); 2,2r-bis (2-aminoethyl)-4-4′-bithiazole (Lee et al.,Biochem. Mol. Biol. Int. 40:151-7, 1996); epilliticine-salen.copperconjugates (Routier et al., Bioconjug. Chem., 8: 789-92, 1997).

E.12. Antiangiogenic Molecules

It is contemplated that in certain embodiments of the invention aprotein that acts as an angiogenesis inhibitor is targeted to a tumor.These agents include, in addition to the anti-angiogenic polypeptidesmentioned above, Marimastat; AG3340; COL-3, BMS-275291, Thalidomide,Endostatin, SU5416, SU6668, EMD121974, 2-methoxyoestradiol,carboxiamidotriazole, CM1O1, pentosan polysulphate, angiopoietin 2(Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, Linomide,thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel,accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470,platelet factor 4 or minocycline.

Thus, in another aspect, the invention relates to a conjugate accordingto the invention for use in medicine. In yet another embodiment, theinvention relates to a pharmaceutical composition comprising a conjugateaccording to the invention and a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Remington'sPharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa.,1995 discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof.

The pharmaceutical compositions of this invention can be administered toa patient by any means known in the art including oral and parenteralroutes. According to such embodiments, inventive compositions may beadministered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

Since CXCR45 is overexpressed in series of tumor cells, the conjugatesof the invention are particularly useful for the delivery of compound ofinterest to said tumor cells. If the active compound has cytostatic orcytotoxic activity, then the conjugates are particularly useful for thetreatment of cancers which over-express CXCR4.

Thus, in another aspect, the invention relates to a conjugate accordingto the invention, a polynucleotide according to the invention, a vectoraccording to the invention or a host cell according to the invention foruse in a method for the treatment of cancer wherein said cancer containscells that expresses CXCR4.

Alternatively, the invention relates to the use of a conjugate accordingto the invention, a polynucleotide according to the invention, a vectoraccording to the invention or a host cell according to the invention forthe manufacture of a medicament for use in a method for the treatment ofcancer wherein said cancer contains cells that expresses CXCR4.

Alternatively, the invention relates to a method for the treatment in asubject of cancer wherein said cancer contains cells that expressesCXCR4 comprising the administration to said subject of a conjugateaccording to the invention, a polynucleotide according to the invention,a vector according to the invention or a host cell according to theinvention.

“Cancer containing cells that expresses CXCR4” include, withoutlimitation, ovarian, bladder, colorectal, lung, head and neck, renal,stomach, uterine, acute lymphoblastic leukemia, and cervical cancers. Ina preferred embodiment, the cancer to be treated using the methodaccording to the present invention is colorectal cancer or pancreaticcancer.

As used herein, the term “colorectal cancer” includes any type of colon,rectal and appendix neoplasia and refers both to early and late adenomasand to carcinomas as well as to the hereditary, familial or sporadiccancer. Hereditary CRC includes those syndromes which include thepresence of polyps, such as the hamartomatous polyposis syndromes andthe most known, familial adenomatous polyposis (FAP) as well asnonpolyposis syndromes such as hereditary nonpolyposis colorectal cancer(HNPCC) or Lynch syndrome I.

In a preferred embodiment of the invention, said colorectal cancer (CRC)is colon cancer, rectal cancer and/or vermiform appendix cancer. Inanother embodiment of the invention, said colorectal cancer is stage 0,stage I, stage II, stage III and/or stage IV colorectal cancer. Thestages of CRC referred to herein correspond to the American JointCommittee on Cancer (AJCC) CRC staging, although other staging methods,such as Dukes and Astler-Coller staging, can equally be used.

As used herein, the phrase “pancreatic cancer” refers to a malignantneoplasm of the pancreas, including but not limited to, adenocarcinomas,adenosquamous carcinomas, signet ring cell carcinomas, hepatoidcarcinomas, colloid carcinomas, undifferentiated carcinomas,undifferentiated carcinomas with osteoclast-like giant cells and isletcell carcinomas.

A “subject”, as used therein, can be a human or non-human animal.Non-human animals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals, as well asreptiles, birds and fish. Preferably, the subject is human.

For example, in the case of treating cancer, an enhanced immune responsecould also be monitored by observing one or more of the followingeffects: (1) inhibition, to some extent, of tumor growth, including, (i)slowing down (ii) inhibiting angiogenesis and (ii) complete growtharrest; (2) reduction in the number of tumor cells; (3) maintainingtumor size; (4) reduction in tumor size; (5) inhibition, including (i)reduction, (ii) slowing down or (iii) complete prevention, of tumor cellinfiltration into peripheral organs; (6) inhibition, including (i)reduction, (ii) slowing down or (iii) complete prevention, ofmetastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion and/or (8) relief, to some extent, of the severity or number ofone or more symptoms associated with the disorder.

The agents are administered in effective amounts. An effective amount isa dosage of the agent sufficient to provide a medically desirableresult. The effective amount will vary with the particular conditionbeing treated, the age and physical condition of the subject beingtreated, the severity of the condition, the duration of the treatment,the nature of the concurrent or combination therapy (if any), thespecific route of administration and like factors within the knowledgeand expertise of the health practitioner. It is preferred generally thata maximum dose be used, that is, the highest safe dose according tosound medical judgment. For example, if the subject has a tumor, aneffective amount may be that amount that reduces the tumor volume orload (as for example determined by imaging the tumor). Effective amountsmay also be assessed by the presence and/or frequency of cancer cells inthe blood or other body fluid or tissue (e.g., a biopsy). If the tumoris impacting the normal functioning of a tissue or organ, then theeffective amount may be assessed by measuring the normal functioning ofthe tissue or organ.

The conjugates are formulated for selected delivery routes including,but are not limited to, topically, intraarticularly, intracisternally,intraocularly, intraventricularly, intrathecally, intravenously,intramuscularly, intratracheally, intraperitoneally and intradermally.

F. Conjugates of the Invention Comprising Antiviral Agents and Usesthereof for the Treatment of Diseases Caused by HIV Infection

Since CXCR4 is expressed predominatly in CD4+ cells, the conjugates ofthe invention are particularly useful for the delivery of compound ofinterest to said CD4+ cells. If the active compound has anti-retroviralactivity or is capable of inducing the death of the cells, theconjugates are particularly useful for the treatment of diseasesassociated with an HIV infection.

Thus, in another aspect, the invention relates to a conjugate accordingto the invention, a polynucleotide according to the invention, a vectoraccording to the invention or a host cell according to the inventionwherein the therapeutic agent is an antiretroviral agent.

The term “antiretroviral agent” as used herein refers to a compound thatinhibits the ability of a retrovirus to effectively infect a host.Antiretroviral agents can inhibit a variety of process including thereplication of viral genetic materials, or entry of retroviruses intocells. In some embodiments, antiretroviral agents are selected from thegroup consisting of: protease inhibitor, a reverse transcriptaseinhibitor, and a viral fusion inhibitor.

In a preferred embodiment, the active compound is an anti-HIV agent thatcan be used for the treatment of a disease associated with HIVinfection. Suitable anti-HIV agents for use as therapeutic agentsaccording to the invention include, without limitation, one or more ofthe following:

-   -   1) Combination drugs: efavirenz, emtricitabine or tenofovir        disoproxil fumarate (ATRIPLA (R)BMS, Gilead); lamivudine or        zidovudine (COMBIVIR (R)/GSK); abacavir or lamivudine (EPZICOM        (R)/GSK); abacavir, lamivudine or zidovudine (TRIZIVIR (R)/GSK);        emtricitabine, tenofovir disoproxil fumarate (TRUVADA        (R)/Gilead).    -   2) Entry and fusion inhibitors: maraviroc (CELSENTRI (R),        SELZENTRY (R)/Pfizer); pentafuside or enfuvirtide (FUZEON        (R)/Roche, Trimeris). In some embodiments, the viral entry        inhibitor is a fusion inhibitor, a CD4 receptor binding        inhibitor, is a CD4 mimic or a gp120 mimic. In some further        embodiments, the viral entry inhibitor is a gp41 antagonist, a        CD4 monoclonal antibody or a CCRS antagonist, including CCRS        antagonist sub-classes such as, for example, zinc finger        inhibitors. In yet another embodiment, the viral entry inhibitor        is a CXCR4 co-receptor antagonist.    -   3) Integrase inhibitors: raltegravir or MK-0518 (ISENTRESS        (R)/Merck).    -   4) Reverse transcriptase inhibitors: Suitable reverse        transcriptase inhibitors for use in the compositions according        to the present invention is one or more compounds selected from        the group consisting of emtricitabine, capravirine, tenofovir,        lamivudine, zalcitabine, delavirdine, nevirapine, didanosine,        stavudine, abacavir, alovudine, zidovudine, racemic        emtricitabine, apricitabine, emivirine, elvucitabine, TMC-278,        DPC-083, amdoxovir, (−)-beta-D-2,6-diamino-purine dioxolane,        MIV-210 (FLG), DFC (dexelvucitabine), dioxolane thymidine,        Calanolide A, etravirine (TMC-125), L697639, atevirdine        (U87201E), MIV-150, GSK-695634, GSK-678248, TMC-278, KP1461,        KP-1212, lodenosine (FddA),        5-[(3,5-dichlorophenyl)thio]-4-isopropyl-1-(4-pyridylmethyl)imidazole-2-methanol        carbamic acid, (−)-I²-D-2,6-diaminopurine dioxolane, AVX-754,        BCH-13520, BMS-56190        ((4S)-6-chloro-4-[(1E)-cyclopropylethenyl]-3,-4-dihydro-4-trifluoromethyl-2        (1H)-quinazolinone), TMC-120, and L697639, where the compounds        are present in amounts effective for treatment of HIV when used        in a combination therapy.    -   5) Protease inhibitors: Suitable protease inhibitors that can be        combined with the miRNAs or polynucleotides encoding miRNAs        according to the invention is selected from the group consisting        of ritonavir, lopinavir, saquinavir, amprenavir, fosamprenavir,        nelfmavir, tipranavir, indinavir, atazanavir, TMC-126,        darunavir, mozenavir (DMP-450), JE-2147 (AG1776), L-756423,        KNI-272, DPC-681, DPC-684, telinavir (SC-52151), BMS 186318,        droxinavir (SC-55389a), DMP-323, KNI-227,        1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine, AG-1859,        RO-033-4649, R-944, DMP-850, DMP-851, and brecanavir (GW640385).        Preferred protease inhibitors for use in combination with a        compound of the present invention include saquinavir, ritonavir,        indinavir, nelfhavir, amprenavir, lopinavir, atazanavir,        darunavir, brecanavir, fosamprenavir, and tipranavir.        Particularly useful such combinations include, for example,        AZT+3TC; TDF+3TC; TDF+FTC; ABC+3TC; and Abacavir+3TC.

Additionally, the compositions according to the present invention mayfurther comprise an antiretroviral agent selected from the groupconsisting of vaccines, gene therapy treatments, cytokines, TATinhibitors, and immunomodulators in amounts effective for treatment ofHIV when used in a combination therapy.

In another aspect, the invention relates to a conjugate according to theinvention, a polynucleotide according to the invention, a vectoraccording to the invention or a host cell according to the inventionwherein the therapeutic agent is selected from the group consisting of

-   -   (i) an antiretroviral agent,    -   (ii) a cytotoxic polypeptide,    -   (iii) a pro-apoptotic polypeptide    -   (iv) a silencing agent and    -   (v) a suicide gene,        for use in the treatment of a disease associated with a HIV        infection.

Alternatively, the invention relates to the use of a conjugate accordingto the invention, a polynucleotide according to the invention, a vectoraccording to the invention wherein the therapeutic agent is selectedfrom the group consisting of

-   -   (i) an antiretroviral agent,    -   (ii) a cytotoxic polypeptide,    -   (iii) a pro-apoptotic polypeptide    -   (iv) a silencing agent and    -   (v) a suicide gene,        for the manufacture of a medicament for use in a method for the        treatment.

Alternatively, the invention relates to a method for the treatment of adisease associated with HIV infection in a subject in need thereof whichcomprises the administration to said subject of a conjugate according tothe invention, a polynucleotide according to the invention, a vectoraccording to the invention wherein the therapeutic agent is selectedfrom the group consisting of

-   -   (i) an antiretroviral agent,    -   (ii) a cytotoxic polypeptide,    -   (iii) a pro-apoptotic polypeptide    -   (iv) a silencing agent and    -   (v) a suicide gene,

The term “retroviral agent” has been defined above.

The terms “cytotoxic polypeptide”, “pro-apoptotic polypeptide”,“silencing agent” and “suicide gene” have been explained in detail abovein the context of the antitumoral agents of the invention and areequally applied in the context of the present methods.

Suitable silencing agents include, without limitation, siRNA constructsfor suppression of HIV replication. These siRNA constructs can bespecific for various HIV targets (reviewed in Morris (2006) Gene Ther13:553-558; Rossi (2006) Biotechniques Supp1:25-29; Nekhai (2006) CurrOpin Mol Ther 8:52-61; and Cullen (2005) AIDS Rev 7:22-25). In order toprevent HIV infection of host T-cells, the invention also featurescomponents to decrease expression of T cell coreceptors (e.g., CCRS andCCR4). Such suppression would be expected to hinder infection of hostT-cells as people with CCR5A32 mutation are resistant to HIV infection.The invention also features the inclusion of multiple siRNA constructs(e.g., constructs against HIV genes and T-cell receptors used for HIVinfection). Here, one siRNA construct can block infection and while asecond siRNA construct prevents progression of infection.

The term “disease associated with HIV infection”, as used herein,includes a state in which the subject has developed AIDS as well as astate in which the subject infected with HIV has not shown any sign orsymptom of the disease. Thus, the compositions of the invention whenadministered to a subject that has no clinical signs of the infectioncan have a preventive activity, since they can prevent the onset of thedisease. The compositions are capable of preventing or slowing theinfection and destruction of healthy CD4+ T cells in such a subject. Italso refers to the prevention and slowing the onset of symptoms of theacquired immunodeficiency disease such as extreme low CD4+ T cell countand repeated infections by opportunistic pathogens such as Mycobacteriaspp., Pneumocystis carinii, and Pneumocystis cryptococcus. Beneficial ordesired clinical results include, but are not limited to, an increase inabsolute naïve CD4+ T-cell count (range 10-3520), an increase in thepercentage of CD4+T-cell over total circulating immune cells (range 1-50percent), and/or an increase in CD4+ T-cell count as a percentage ofnormal CD4+ T-cell count in an uninfected subject (range 1-161 percent).“Treatment” can also mean prolonging survival of the infected subject ascompared to expected survival if the subject did not receive any HIVtargeted treatment.

The present invention further relates to preventing or reducing symptomsassociated with HIV infection. These include symptoms associated withthe minor symptomatic phase of HIV infection, including, for example,shingles, skin rash and nail infections, mouth sores, recurrent nose andthroat infection and weight loss. In addition, further symptomsassociated with the major symptomatic phase of HIV infection, include,for instance, oral and vaginal thrush (Candida), persistent diarrhea,weight loss, persistent cough and reactivated tuberculosis or recurrentherpes infections, such as cold sores (herpes simplex). Other symptomsof full-blown AIDS which can be treated in accordance with the presentinvention include, for instance, diarrhea, nausea and vomiting, thrushand mouth sores, persistent, recurrent vaginal infections and cervicalcancer, persistent generalized lymphadenopathy (PGL), severe skininfections, warts and ringworm, respiratory infections, pneumonia,especially Pneumocystis carinii pneumonia (PCP), herpes zoster (orshingles), nervous system problems, such as pains, numbness or “pins andneedles” in the hands and feet, neurological abnormalities, Kaposi'ssarcoma, lymphoma, tuberculosis or other similar opportunisticinfections.

Preparation of the Conjugates of the Invention

Methods for the preparation of the conjugates are provided. Thesemethods include chemical conjugation methods and methods that rely onrecombinant production of the conjugates.

The targeting peptide used in the practice of the invention may beattached to the active compound of therapeutic or diagnostic interest bychemical modification. Typically, the chemical methods rely onderivatization of the active agent (therapeutic or diagnostic) with thedesired linking agent, and then reaction with the targeting peptide. Thechemical methods of derivatization may be carried out using bifunctionalcross-linking agents.

In practicing the chemical method, a targeting peptide that is producedby any means, typically by expression of DNA in a bacterial oreukaryotic host or by chemical synthesis is chemically coupled with theactive agent. If the targeting peptide or active agent does not containsuitable moieties for effecting chemical linkage it can be derivatized.For example, the agent, such as Shiga toxin, gelonin or other suchagent, can be derivatized such as by reaction with a linking agent, suchas N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP). In otherembodiments, the targeted agent, such as shiga A chain, is modified ator near the N-terminus to include a cysteine residue, so that theresulting modified agent can react with the chemokine receptor-bindingmoiety protein without further derivatization.

Non-limiting examples of possible chemical groups involved in such theconjugation are: a carboxylic acid group on the targeting peptide, whichcould be reacted with an amino group on the active agent (for example,the .epsilon.-amino group on a lysine side chain) to form an amide grouplinking the targeting peptide and the active agent; an amino group onthe targeting peptide, which could be reacted with a carboxylic acidgroup on the active agent (for example, the carboxylic acid group on aglutamate or aspartate side chain) to form an amide group linking thetargeting peptide and the active agent; a disulfide group on thetargeting peptide, which could be reacted with a thiol group on theactive agent (for example, the thiol group on a cysteine side chain) toform a disulfide group linking the targeting peptide and the activeagent.

Once conjugated, the conjugate generally will be purified to separatethe conjugate from unconjugated targeting agents or from othercontaminants. A large number of purification techniques are availablefor use in providing conjugates of a sufficient degree of purity torender them clinically useful. Purification methods based upon sizeseparation, such as gel filtration, gel permeation or high performanceliquid chromatography, will generally be of most use. Otherchromatographic techniques, such as Blue-Sepharose separation, may alsobe used.

In certain embodiments, the conjugate is a fusion protein of thetargeting peptide and the therapeutic agent, in which case the fusionprotein or peptide may be isolated or purified. Protein purificationtechniques are well known to those of skill in the art. These techniquesinvolve, at one level, the homogenization and crude fractionation of thecells, tissue or organ to polypeptide and non-polypeptide fractions. Theprotein or polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, gel exclusion chromatography,polyacrylamide gel electrophoresis, affinity chromatography,immunoaffmity chromatography and isoelectric focusing. A particularlyefficient method of purifying peptides is fast performance liquidchromatography (FPLC) or even high performance liquid chromatography(HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50 percent, about 60 percent, about 70 percent, about80 percent, about 90 percent, about 95 percent, or more of the proteinsin the composition.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

The invention is described by way of the following examples which are tobe construed as merely illustrative and not limitative of the scope ofthe invention.

EXAMPLES

Materials and Methods

Protein Production and Purification.

GFP fusion genes were produced by standard recombinant DNA technologies,to encode hybrid proteins containing a His tag at the C terminus and arelevant CXCR4 ligand at the amino terminus. The encoded proteinsT22-GFP-H6, V1-GFP-H6, vCCL2-GFP-H6 and CXCL12-GFP-H6 (Table 2) wereproduced in soluble form in Escherichia coli strain Origami B (OmpT-,lon-, trxB-, gor-(Novagen)). Bacteria carrying the different pET22bversions (Novagen 69744-3) encoding for each fusion proteins, were grownin Luria-Bertani (LB) medium and protein expression was induced by 0.1mM isopropyl-β-D-thiogalactopyronaside (IPTG). Bacterial cultures werecentrifuged and resuspended in buffer A (20 mM Tris-HCl pH 7.5, 500 mMNaCl, 10 mM Imidazole) in presence of EDTA-free protease inhibitor(Complete EDTA-Free; Roche). The cells were then disrupted by 1100 psipresion using a french press (Thermo FA-078A).

Proteins were purified from crude cell extracts by 6×His tag affinitychromatography using HiTrap Chelating HP 1 ml Ni²⁺ columns (GEhealthcare) in an AKTA purifier FPLC (GE healthcare). Positive fractionswere collected in elution buffer (Tris-HCl 20 mM pH 7.5, 500 mM NaCl,500 mM Imidazole), dialyzed against PBS+10% glycerol or 20 mM Tris 500mM NaCl buffers, and quantified by Bradford's procedure. Proteinintegrity was characterized by mass spectrometry (MALDI-TOF) andN-terminal sequencing.

DNA Retardation Assays and Transmission Electron Microscopy

DNA-protein incubation and DNA mobility assays were performed accordingto previously published protocols (2) in PBS pH 6+10% glycerol.

For transmission electron microscopy, purified proteins were diluted to0.2 mg/ml final concentration. All the protein samples were negativelystained with 2% Uranyl acetate onto carbon coated grids. The sampleswere also platinated by depositing evaporated 1 nm platinum layer overthe sample containing grids. All the samples were visualized in HitachiH-7000 transmission electron microscope.

Cell Culture and Confocal Laser Scanning Microscopy

HeLa (ATCC-CCL-2) cell line was used in all the experiments andmonitored in vivo, in absence of fixation. Cells were maintained in MEM(GIBCO, Rockville, Md.) supplemented with 10% Fetal Calf Serum (GIBCO)and incubated at 37° C. and 5% CO₂ in a humidified atmosphere. GFPfusion proteins at the concentrations indicated, were added to cellculture in the presence of Optipro medium (GIBCO, Invitrogen) 20 hbefore confocal analysis, except for time-course studies and studies ofinternalization in the presence of serum (complete media). For confocalanalysis, cells were grown on Mat-Teck culture dishes (Mat Teck Corp.,Ashland, Mass., United States). Nuclei were labelled with 20 pg/m1Hoechst 33342 (Molecular Probes, Eugene, Oreg., United States) and theplasma membrane was labelled with 2.5 μg/ml CELLMASK Deep Red (MolecularProbes, Invitrogen, Carlsbad, Calif., USA) for 5 min in the dark. Cellswere washed in PBS (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Livecells were recorded with a TCS-SP5 confocal laser scanning microscope(Leica Microsystems, Heidelberg, Germany) using a Plan Apo 63x/1.4 (oilHC×PL APO lambda blue) objective. Hoechst 33342 DNA labels was excitedwith a blue diode (405 nm) and detected in the 415-460 nm range.GFP-proteins were excited with an Ar laser (488 nm) and detected in the525-545 nm range. CELLMASK™ was excited with a HeNe laser (633 nm) anddetected in the 650-775 nm range. To determine the protein localizationinside the cell, stacks of 10 to 20 sections every 0.5 μm along the cellthickness were collected. The projections of the series obtained weregenerated with Leica LAS AF software, and three-dimensional models weregenerated using Imaris v. 6.1.0 software (Bitplane; Zürich,Switzerland).

Protein-Mediated Plasmid Transfection

For expression experiments, 20, 40, 60 and 80 μg of T22-GFP-H6 protein(1, 2, 3 and 4 retardation units —RU—) and 1 μg of TdTomato expressionvector were mixed into a final volume of 85 μl of PBS+10% glycerolbuffer, and complexes were formed after 1 h at room temperature, afterwhich convenient volumes of Optipro were added. The complex was gentlyadded to HeLa cells, followed by incubation for 24 and 48 h at 37° C. in5% CO₂ atmosphere. TdTomato gene expression was monitored by flowcytometry and by confocal microscopy. Cells without treatment, or justincubated with the expression vector or the protein alone, were used ascontrols.

Flow Cytometry

Cell samples were analyzed after treatment with 0.5 mg/ml trypsin, 4Nain HBSS for 1 min on a FACSCanto system (Becton Dickinson), using a 15mW air-cooled argon-ion laser at 488 nm excitation. Fluorescenceemission was measured with detector D (530/30 nm band pass filter) forEGFP and detector C (585/42 nm band pass filter) for TdTomatofluorescent protein. To test the effect of different published trypsintreatments (Lundberg, 2003, Mol. Ther., 8:143 and Egorova et al., 2009,J. Gene Med.,11:772) in our internalization results, we used 0.5 mg/mland 1 mg/ml of trypsin with or without 1 M NaCl for 1 or 15 min beforecytometry analysis.

Competition Assay

To assess the binding specificity of T22-GFP-H6 to CXCR4, HeLa cellswere grown in a 24-well plaque at 70% confluence in a competition assay.Cells were pre-incubated for 30 min with increasing amounts of thenatural CXCR4 ligand SDF1alpha in Optipro, to reach 25 nM and 250 nM, orwith the irrelevant proteins GFP-H6 and human alpha-galactosidase (bothat 250 nM). Then, T22-GFP-H6 was added to the cultures at 25 nM andfurther incubated. One hour later, cells were treated with 1 mg/mLtrypsin for 15 min and intracellular fluorescence determined by flowcytometry. Data were analyzed with WinMDI, Microsoft Excel 2003 andSigmaplot 10 software.

Example 1

The Peptide T22 is Able to Promote the Internalization of Functional andSoluble Fused Proteins into Target CXCR4+ Cells.

In this study, the internalization properties of four peptides, alreadydescribed as CXCR4 ligands, namely T22, V1, vCCL2, CXCL12 (see Table 1)was investigated. This was done by constructing four model proteins inwhich these peptides had been fused to GFP-H6 (Table 2), rendering fullyfluorescent proteins. When exposing cultured HeLa cells to theseconstructs, those in contact with T22-GFP-H6 were dramatically labelledwith green fluorescence over the rest of peptides (FIGS. 1A, 1B and 2A,2B, 2C, and 2D). This uptake was concentration dependent (see FIG. 3A)and observed even after treating the cells with trypsin after thebinding of the fusion proteins to eliminate superficially attachedprotein (FIG. 3B). To assess that the signal was due to actual proteinuptake, cultured cells were submitted to confocal analysis thatconfirmed the highly efficient internalization of T22-GFP-H6, and thepoor penetrability of the rest of tested peptides (FIGS. 3C, 3D, 3E, 3F,3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, and 3O).

Thus, the peptide T22 is able to promote the internalization offunctional and soluble fused proteins into target CXCR4+ cells.

Example 2

The Peptide T22 is Able to Promote the Internalization and Expression ofPlasmid DNA into Target CXCR4+ Cells.

Different biological and physicochemical features of T22-GFP-H6 weredetermined by alternative procedures (FIGS. 3C, 3D, 3E, 3F, 3G, 3H, 3I,3J, 3K, 3L, 3M, 3N, and 3O). Among them, it was noteworthy theperinuclear localization of T22-GFP-H6 and the presumed docking of theprotein in the nuclear membrane (FIGS. 3F, 3G and 3H, see the inset).This suggested that apart from the potential of T22 in the delivery offunctional proteins (in the model a fully fluorescent GFP shown inExample 1), this peptide could also deliver expressible DNA andeventually promote their entrance into the nuclear compartment. Toexplore this possibility, it was first determined whether T22-GFP-H6could bind plasmid DNA in vitro, what was fully proved by DNAretardation assays (FIGS. 4A, 4B, 4C and 4D). Furthermore, the potentialgene expression of the reporter TdTomato gene, encoding a redfluorescent protein, was analyzed when this gene was associated toT22-GFP-H6 complexes. The analysis of transfected HeLa cells (FIGS. 4B,4C and 4D) indicated that the reporter gene is expressed, and notmatching red (DNA expression) and green (T22-GFP-H6) fluorescence wereclearly observed in those cultured cells.

Example 3

The Internalization of T22-GFP-H6 is Inhibited by a Natural Ligand ofCXCR4

The ability of a natural CXCR4 ligand (SDFalpha) to inhibit theinternalization of T22-GFP-H6 was tested by contacting CXCR4+ cells withthe T22-GFP-H6 in the presence of the protein SDFalpha, at differentT22-GFP-H6/SDFalpha ratios (1:0, 1:1, 1:10). Two irrelevant proteins,GFP-H6 and GLA were used as controls at a 1:10 ratio. The results showedthat SDFalpha was capable to inhibit in a dose-dependent manner theinternalization of the T22-GFP-H6, whereas no effect was observed withthe unrelated proteins (FIG. 5 ).

Example 4

Selective Biodistribution of the T22-GFP In Vivo to Primary Tumors

A fusion protein comprising T22 as a target moiety (which specificallybinds to the CXCR4 receptor) and a green fluorescent protein wasadministered to immunosuppressed mice containing a primary tumorresulting from the xenotransplant of CXCR4+ SW1417 human colorectalcancer cells in the cecum of the mice. Distribution of T22-GFP wasdetermined at several time points (5, 24 or 48 h) after intravenoussingle dose administration of a dose range varying form 20 to 500 ug.T22-GFP accumulated 8-47 fold with respect to control animals, asmeasured by fluorescence quantitation, using a IVIS200 system (Xenogen).No fluorescence is detected in primary tumor tissue in control micetreated with vehicle.

Example 5

Selective Biodistribution of the T22-GFP to the Peritoneal Metastasesand Mesenteric and Diaphragmatic Lymph Nodes

An intravenous single dose of 500 μg of T22-GFP was administered to miceshowing peritoneal metastases and mesenteric and diaphragmatic lymphnodes derived from CXCR4+ SW1417 human colorectal cancer cellsxenotransplanted in the cecum of immunosuppressed mice. T22-GFPaccumulated 20-40 fold in peritoneal metastatic foci, as measured byfluorescence quantitation using a IVIS200 system (Xenogen) at 5 h or 24h after. No fluorescence was detected in the liver parenchyma,pancreatic parenchyma, kidney, heart, non-tumor intestine, non-tumorlymph nodes, lung or spleen of the experimental or control (vehiclecontrol) animal. Whereas accumulation of the T22-GFP fusion protein wasobserved in tumor tissues, no accumulation of T22-GFP was observed innormal tissues. Fluorescence was observed in the billiary vesicle(attached to the liver) or in the pancreas in both experimental andcontrol animals, which could be attributed to fluorescent proteinssecreted by these organs.

TABLE 1Main physicochemical properties of the peptides used in this study.Peptide Sequence mer Mw (Da) P N Arg pI AI H SI T22MRRWCYRKCYKGYCYRKCR (SEQ ID 19 2623.1  8 0 5 9.96  0 -1.516 39.06NO: 28) (stable) V1 MLGASWHRPDKCCLGYQKRPLP (SEQ 22 2557  4 1 2 9.3857.73 -0.705 55.14 ID NO: 29) (unstable) vCCL2MLGASWHRPDKCCLGYQKRPLPQVLLSSW 71 8103.6 12 3 4 9.9 79.58 -0.361 37.48YPTSQLCSKPGVIFLTKRGRQVCADKSKD (stable) WVKKLMQQLPVTA (SEQ ID NO: 30)CXCL12 MKPVSLSYRCPCRFFESHVARANVKHLKI 68 7966.4 12 4 5 9.81 97.5 -0.32816.24 LNTPNCALQIVARLKNNNRQVCIDPKLKW (stable) IQEYLEKALN (SEQ ID NO: 31)P: Positively charged amino acids; N: Negatively charged amino acids;Arg: Number of arginine residues; pI: Isolelectric point; Al: Aliphaticindex; H: Hidrophobicity; SI: Stability index;

TABLE 2Main physicochemical properties of the fusion proteins generated in this study.SF Fusion Mw S (FU/μg Protein mer (Da) (%) protein) P N pI AI H SI TASHphoAS HphiAS NCP NCT T22-GFP- 269 30691.5 100 13.76 (PBS) 36 34 8.1266.99 -0.71 34.18 16039 8828 7212  3  0 H6 22.8 (Tris) (stable) (SEQ IDNO: 33) V1-GFP- 272 30625.4 100 12.01 (PBS) 32 35 6.62 70.92 -0.65335.85 14787 8405 6382 -4 -6 H6 12.44 (Tris) (stable) (SEQ ID NO: 34)vCCL2- 321 36172 100 17.15 (PBS) 40 37 8.38 73.74 -0.585 34.89 2478015428 9353  2  1 GFP-H6 11.57 (Tris) (stable) (SEQ ID NO: 35) CXCL12-318 36034.8   5.33 27.4 (PBS) 40 38 8.11 77.52 -0.58 29.85 17806 100077799  0  0 GFP-H6 14.32 (Tris) (stable) (SEQ ID NO: 36) S: Solubility;SF: Specific fluorescence; P: Positively charged amino acids; N:Negatively charged amino acids; pI: Isolelectric point; AI: Aliphaticindex; H: Hidrophobicity; SI: Stability index; TAS: Total accessibilityto solvent; HphoAS: Hydrophobic accessibility to solvent; HphiAS:Hydrophilic accessibility to solvent; NCP: Net charge in PBS (pH = 7.4);NCT: Net charge in Tris (pH = 8)

The invention claimed is:
 1. A conjugate comprising (i) a targetingpeptide comprising the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 5) or apeptide with at least one conservative amino acid substitution in thesequence SEQ ID NO: 5, wherein the conservative amino acid substitutionis selected from the following six groups each contain amino acids thatare conservative substitutions for one another: 1Alanine (A), Serine(S); Threonine (T); 2Aspartic acid (D), Glutamic acid (E); 3Asparagine(N), Glutamine (Q); 4Arginine (R), Lysine (K); 5Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6Phenylalanine (F), Tyrosine (Y),Tryptophan (W), and (ii) a therapeutic agent, wherein the targetingpeptide specifically binds to chemokine receptor type 4 (CXCR4) andpromotes internalization of the therapeutic agent in a cell expressingCXCR4 and wherein the therapeutic agent is a polypeptide or protein. 2.The conjugate according to claim 1 wherein the targeting peptideconsists of the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 5).
 3. Theconjugate according to claim 1 wherein the therapeutic agent is apolypeptide.
 4. The conjugate according to claim 3 wherein thetherapeutic agent and the targeting peptide are part of a fusionprotein.
 5. The conjugate according in claim 1, wherein the therapeuticagent is provided within a nanotransporter and wherein thenanotransporter is coupled to the targeting peptide.
 6. The conjugateaccording to claim 1, wherein the therapeutic agent is an antitumoragent.
 7. The conjugate according to claim 6 wherein the antitumor agentis selected from the group consisting of (i) a cytotoxic polypeptide,(ii) an antiangiogenic polypeptide, (iii) a polypeptide encoded by atumor suppressor gene, (iv) a pro-apoptotic polypeptide, (v) apolypeptide having anti-metastatic activity, and (vi) a polypeptideencoded by a polynucleotide which is capable of activating the immuneresponse towards a tumor.
 8. The conjugate according to claim 1, whereinthe therapeutic agent is an antiretroviral agent.
 9. The conjugateaccording to claim 1, wherein the therapeutic agent is selected from thegroup consisting of (i) an antiretroviral agent, (ii) a cytotoxicpolypeptide, and (iii) a pro-apoptotic polypeptide.
 10. A method for thetreatment of cancer in a subject in need thereof wherein said cancercontains cells that express CXCR4 said method comprising theadministration of a conjugate according to claim
 7. 11. The methodaccording to claim 10 wherein the cancer is pancreatic or colorectalcancer.